functional characterisation of two channels proteins involved in
TRANSCRIPT
Functional Characterisation of Two Channels Proteins
Involved in Leguminous Symbiosis
Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
vorgelegt von
Charpentier Myriam
München
im September 2008
2
Dekan: Prof. Dr. Jürgen Soll
Gutachter: Prof. Dr. Martin Parniske
Gutachter: Prof. Dr. Heinrich Jung
Datum der Disputation: 18.12.2008
3
To my father (194 9- 199 9 )
4
Table of contents
List of abbreviations 7
List of Units 9
Abstract 10
Zusammenfassung 11
1. Introduction 12
1.1 Endosymbiotic plant-microbe interactions 12
1.2 Root-nodule-symbiosis early ion fluxes 17
1.3 Dissection of the calcium-spiking pathway 22
1.4 Castor and pollux mutant and aim of the study 23
2. Results 28
2.1 CASTOR and POLLUX identification 28
2.2 Sequence analyses 31
2.3 Nuclear localization of CASTOR and POLLUX 35
2.4 Homodomerization of CASTOR and POLLUX 40
2.5 Expression of POLLUX and DMI1 under the control of P35S restore the nodulation
phenotype of castor-12 mutant 43
2.6 Functional characterization 47
2.6.1 Yeast complementation assays 47
2.6.1.1 cch1Δmid1Δ and trk1Δ yeast mutants complementation assays 47
2.6.1.2 MAB2d yeast mutant complementation assays 48
2.6.2 Electrophysiological analysis 50
2.6.2.1 CASTOR is a cation channel 50
2.6.2.2 Magnesium mediates voltage-dependent blockage of CASTOR 54
2.6.2.3 CASTOR sensitivity to putative binding ligands 55
2.7 CASTOR interacting component 57
2.7.1 Yeast-two-hybrid assay with common symbiotic components 58
2.7.2 Screening of a L. japonicus roots cDNA libraries using yeast-two-hybrid system 59
2.7.2.1 Root nodule phenotype of transformed L. japonicus roots expressing
RNAiLjSNF7 construct 61
2.7.2.2 Sub-cellular localization of LjSNF7 62
5
3. Discussion 64
3.1 Nuclear localization of CASTOR and POLLUX 64
3.2 CASTOR and POLLUX are non-selective cation channels 65
3.3 Channel gating 67
3.4 Hypothetical role of LjSNF7 in the root nodule symbiosis 68
3.5 CASTOR and POLLUX are required for calcium spiking 70
4. Material and methods 73
4.1 Material 73
4.1.1 Plants and chemicals 73
4.1.2 Enzymes and kits 73
4.1.3 Strains, oligonucleotides, vectors and clones 73
4.1.4 Antibodies 74
4.2 Methods 76
4.2.1 Bioinformatics 76
4.2.2 Genetics methods 76
4.2.3 Molecular biological methods 77
4.2.3.1 General molecular biological methods 77
4.2.3.2 TILLING 77
4.2.3.3 Cloning strategies 78
4.2.3.4 RNA isolation 78
4.2.3.5 Reverse transcription (RT)-PCR, Rapid Amplification of cDNA ends (RACE)-PCR
and quantitative Reverse Transcription (qRT)-PCR 78
4.2.3.6 Site directed mutagenesis 79
4.2.3.7 Yeast transformation methods 79
4.2.4 Biochemical methods 79
4.2.4.1 General biochemical methods 79
4.2.4.2 Protein extraction from L. japonicus root 79
4.2.4.3 Protein extraction from N. benthamiana leaves 80
4.2.4.4 Protein extraction from S. cerevisiae 80
4.2.4.5 In vitro expression and purification 80
4.2.5 Electrophysiological methods 81
4.2.6 Plant transformation methods 82
6
4.2.6.1 A. tumefaciens-mediated transient transformation 82
4.2.6.2 A. rhizogenes-mediated transient transformation 82
4.2.7 Cell biological methods 83
4.2.7.1 L. japonicus cell culture protoplast transfection 83
4.2.8 Histochemical methods 84
4.2.8.1 Immunogold electron microscopy 85
4.2.8.2 GUS assay 85
4.2.8.3 Mycorrhiza staining 85
4.2.9 Fungal and bacterial inoculation methods 85
4.2.9.1 Inoculation with G. intraradices 85
4.2.9.2 Inoculation with M. loti 85
5. References 86
6. Acknowledgement 102
7. Appendix 103
7.1 List of publication 103
7.1.1 Papers 103
7.1.2 Posters and conferences 103
7.1.3 Talks 104
7.2 List of figures 105
7.3 List of tables 106
7.4 Erklärung 107
7.5 Curriculum vitae 108
7
List of abbreviations
a.a. Amino acid AFLP Amplified fragment length polymorphism AM Arbuscular mycorrhiza AS Alternative spliced BAC Bacterial artificial chromosomes BiFC Bimolecular fluorescence complementation bp Base pair Ca2+ Calcium CaCl2 Calcium chloride cADP-rib 1-(5-phospho-b-D-ribosyl) adenosine 5-phosphate cyclic anhydride CAPS Cleaved amplified polymorphic sequence CDD Conserved domain database cDNA Complementary deoxyribonucleic acid CODDLE Codons Optimized to Discovered Deleterious Lesions DNA Deoxyribonucleic acid DGPP Diacylglycerol pyrophosphate DMI Does not make infection DsRed Discosoma sp. red fluorescent protein DTT Dithiothreitol DUF Domain of unknown function EDTA Ethylenediaminetetraacetic acid EMS Ethyl methane-sulfonate ENOD Early nodulation ER Endoplasmic reticulum Erev Reverse potential ESCRT Endosomal sorting complex required for transport EST Expressed sequence tags F Faraday constant; Nqe = 9.6485 x 104 C mol-1 FNR Ferredoxin NADP(H) oxidoreductase GFP Green fluorescent protein GUS β-glucuronidase HRP Horseradich peroxidase INM Inner nuclear membrane InsP3 Inositol triphosphate IPD Interacting protein of DMI3 IT Infection thread ITD Infection thread deficient K+ Potassium kb Kilobase KCl Potassium chloride Li+ Lithium MtPT4 Medicago truncatula phosphate transporter MEGA-9 N-Nonanoyl-N-methylglucamine Na+ Sodium NaCl Sodium chloride
8
NAADP Nicotinic acid adenine dinucleotide phosphate NAD+ Nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate NAD(P)bd Nicotinamide adenine dinucleotide phosphate binding domain NCBI National Centre for Biotechnology Information NE Nuclear envelope NFR Nod factor receptor NLS Nuclear localization signal nt nucleotide NUP Nucleoporin ONM Outer nuclear membrane PA Phosphatidic acid PCR Polymerase chain reaction PLD Phospholipase D PHYLIP Phylogeny inference package PPA Pre-penetration apparatus PI-PLC Phosphoinositide-dependent phospholipase C PIP2 Phosphatidylinositol 4,5-bisphosphate PIT Pre-infection thread PTC Premature termination codon QAs Quaternary ammonium ions qRT Quantitative Reverse Transcription R Molar gas constant; 1.987 cal mol-1K-1 RACE Rapid Amplification of cDNA ends RFP Red fluorescent protein RNAi Ribonucleic acid interference RNS Root nodule symbiosis SINA Seven in absentia SD Yeast synthetic defined medium SDS Sodium dodecyl sulfate SNF7p sucrose non-fermenting protein 7 SSR Simple sequence repeat SYMRK Symbiosis receptor-like kinase T Thermodynamic temperature TAC Transformable bacterial artificial chromosomes TBA Tetrabutylammonium TILLING Targeted induced local lesions in genomes Tm Annealing temperature TP Transit peptide v/v By volume YFP Yellow fluorescent protein
9
List of Units A Ampere C Celsius Da Dalton g Times gravity L Liter m Meter M mol/L ODx Optical density of an element for a given wavelength x pH “Power of hydrogen” or measure of the activity of dissolved
hydrogen ions rpm Centrifuge rotor speed s second S Siemens V Volt
10
Abstract
Legume-rhizobial symbiosis results in the formation of a new organ, the nitrogen-fixing
root nodule. A chemical communication between both partners accompanies the invasion
of plant host cells by bacteria and the development of the root nodule. In response to
plant-released flavonoids, rhizobia produce lipo-chito-oligosaccharide signalling
molecules, so called Nod factors. Early signal transduction in legumes such as Lotus
japonicus and Medicago truncatula, is associated with a succession of tightly
orchestrated ion fluxes across different membrane systems of the host cell. The Nod
factor perception at the plasma membrane triggers Ca2+ oscillations that are associated
with the nucleus. CASTOR and POLLUX are required for Ca2+ spiking. Homology
modeling suggested CASTOR and POLLUX might be ion channels. However,
experimental confirmation was lacking. Therefore we performed biochemical and
electrophysiological analysis to define their role. Here we show that CASTOR and
POLLUX form two independent homocomplexes in the nuclear rim in planta. We
reconstituted CASTOR in planar lipid bilayers and electrophysiological measurements
revealed that CASTOR is a cation channel preferentially permeable to potassium. The
permeability of the sequence-related POLLUX for cation could be as well demonstrated
through expression of POLLUX in different yeast mutants. Furthermore, we demonstrate
that a voltage-dependent magnesium blocking mechanism contributes to reduce the
conductance of CASTOR at negative membrane potential. By screening a L. japonicus
roots cDNA library using yeast-two-hybrid system, a SNF7 protein interacting with
CASTOR was found which acts as positive regulator in the nodulation pathway.
Collectively the data demonstrate that both CASTOR and POLLUX are nuclear localized
cation channels. Therefore, we propose that CASTOR and POLLUX may act as counter
ion channels to facilitate a rapid efflux of charge associated with the calcium efflux.
Alternatively and not mutually exclusive, they may catalyze a nuclear membrane
depolarization leading to the activation of calcium channels responsible for calcium
spiking.
11
Zusammenfassung
Leguminosen können mit Bakterien, den so genannten Rhizobien, eine Symbiose
eingehen, wobei in neu gebildeten Pflanzenorganen, den Wurzelknöllchen, molekularer
Stickstoff aus der Luft fixiert wird. Das Eindringen der Bakterien in die Pflanzenzellen
und die darauf folgende Entstehung der Wurzelknöllchen sind Folge eines molekularen
Signalaustauschs zwischen beiden Partnern. Als Reaktion auf Flavonoide, die von der
Pflanze ausgeschüttet werden, produzieren Rhizobien Lipochitooligosaccharide, die so
genannten Nod-Faktoren. Perzeption dieser bakteriellen Signalmoleküle in Leguminosen
wie Lotus japonicus und Medicago truncatula induziert nukleäre Kalzium-Oszillationen,
die die Funktion von zwei Proteinen, CASTOR und POLLUX, erfordern.
Strukturvorhersagen, die auf Sequenzvergleichen mit bereits bekannten Proteinen
basieren, legen die Vermutung nahe, dass es sich bei CASTOR und POLLUX um
Ionenkanäle handelt. Ein experimenteller Nachweis fehlte jedoch. Daher wurden in dieser
Arbeit CASTOR und POLLUX mit biochemischen und elektrophysiologischen
Methoden analysiert, um ihre genaue Funktion zu identifizieren. Es konnte gezeigt
werden, dass CASTOR und POLLUX in planta zwei voneinander unabhängige Homo-
Komplexe in der Kernmembran bilden. Wir rekonstituierten CASTOR in planaren
Lipiddoppelschichten für elektrophysiologische Messungen, die ergaben, dass CASTOR
ein kationenpermeabler Ionenkanal mit einer schwachen Präferenz für Kalium ist. Die
Permeabilität für Kalium von POLLUX, dessen Sequenz mit der von CASTOR verwandt
ist, wurde durch die Expression von POLLUX in einer Hefe-Mutante mit defektem
Kaliumtransport bestätigt. Anhand einer ‚Yeast-two-hybrid’-Sichtung einer L. japonicus
cDNA-Bibliothek, wurde ein SNF7 Protein identifiziert, das mit CASTOR interagiert und
ein positiver Regulator der Signaltransduktionskette der Wurzelknöllchensymbiose ist.
Die in dieser Arbeit gewonnenen Daten belegen, dass CASTOR und POLLUX
Kationenkanäle in der Kernmembran sind, die den schnellen Efflux von Kalzium durch
einen gegensätzlichen Ladungsausgleich ermöglichen. Eine alternative oder zusätzliche
Funktion der beiden Proteine könnte die Depolarisierung der Kernmembran darstellen,
die für die Aktivierung der Kalziumkanäle und somit für die Kalzium-Oszillationen
verantwortlich ist.
12
1. Introduction
1.1 Endosymbiotic plant-microbe interactions
In the mid-nineteenth century, de Bary defined the term “symbiosis” (from the Greek:
sym = together, bio = life) as a close physical association between two organisms of
different species. This definition which could be applied to a wide range of biological
interaction, was then narrowed by Pierre-Joseph van Benedén who restricted as
symbiotic, a mutually beneficial relationships between two organisms (Wilkinson, 2001).
Most land plants rely on symbiosis with soil microorganisms such as fungi and bacteria
to provide them with inorganic compounds and trace elements. Among microorganisms,
some symbionts accommodate within the host plants cells to form an endosymbiosis
(endo = inner).
The arbuscular mycorrhiza (AM), endomycorrhiza formed by fungi of the phylum
Glomeromycota and the majority of land plants, is one of the most significant plant-
fungal endosymbiosis (Schüssler, 2001; Trappe, 1987). In this mutually beneficial
association, AM fungi provide the plants with phosphate and other mineral nutrients and
in return obtain carbohydrates from their hosts (Hodge et al., 2001). The fossil record for
existence of AM, 450 million years ago, demonstrates that the AM is as old as the earliest
land plants (Remy et al., 1994). In comparison, root nodule symbiosis (RNS),
endosymbiosis between soil nitrogen-fixing bacteria and plants belonging to a clade
within the Eurosid, including the Fabales, Fagales, Cucurbitales and Rosales, is more
recent; the oldest fossil nodule-like structures are dated at 65 million years (Herendeen et
al., 1999; Soltis et al., 2000). RNS occurs with two different types of soil bacteria: Gram-
positive bacteria of the genus Frankia and members of Gram-negative bacteria, termed
rhizobia. Plants of the Fabales, Cucurbitales and Rosales nodulate with gram-positive
actinobacteria Frankia (Swensen and Benson, 2008). In contrast, with the exception of
the genus Paranosporia in the elm family, the rhizobia, including the Rhizobium,
Bradyrhizobium, Azorhizobium, Mesorhizobium and Sinorhizobium, associate with plants
of exclusively one order, the Fabales (Doyle, 1998; Marvel et al., 1987).
13
The phenotypic analysis of legume mutants impaired in both RNS and AM
revealed a genetic link between both symbioses (Catoira et al., 2000; Kistner et al., 2005).
From the existence of this common genetic program the hypothesis arises that pre-
existing AM genetic program has been recruited to serve the bacterial endosymbiosis
development (Albrecht et al., 1999; LaRue and Weeden, 1994). For both AM and
rhizobia RNS, the success of the relationship between the symbionts and the plants
depend on four major steps: recognition, penetration, proliferation and nutrient transfer.
During these steps, structural and physiological similarity can be observed between the
two endosymbioses.
In the case of the interaction between the AM fungi and the plant host, the
signalling events responsible for mutual recognition are not fully understood. Spores of
AM fungi can germinate spontaneously without plant-derived signals (Juge et al., 2002).
Nevertheless, depending on the fungi/host interaction, flavonoids-containing roots
exudates can enhance or decrease the spore germination, indicating that those plant-
phenolics are perceived by the fungi (Nair et al., 1991; Vierheilig et al., 1998). The spore
germination can also be enhanced by the host plant-derived strigolactone. The
strigolactone, newly identified as endogenous plant hormones (Umehara et al., 2008), has
been shown to induce hyphal branching and metabolic activity of the fungi (Akiyama et
al., 2005; Besserer et al., 2006; Buee et al., 2000). In addition to the growth and
metabolic changes, the fungi respond to the plant by releasing signalling molecules which
are key messengers to alert the plant of the symbiont presence and trigger the plant
symbiosis program. The first evidence for fungal signalling molecules, so-called “Myc
factors”, was gained from experiments using a Medicago truncatula EARLY
NODULATION11 promoter (PENOD11): β-glucuronidase (GUS) fusion, which is induced
in response to both AM fungi and rhizobia (Journet et al., 2001). The ENOD11 gene
encodes a repetitive Pro-rich protein, believed to be a functional component of the plant
extracellular matrix and induced upon AM and RNS (Journet et al., 2001). The AM
branching hyphae, localized in the vicinity of the host root but physically separated by a
permeable cellophane membrane, induced GUS expression in epidermal and cortical
roots cells (Kosuta et al., 2003). This experiment indicates that the host roots perceive the
diffusible “Myc factor(s)”. Multiple experiments confirmed the presence of diffusible
fungal factor(s) (Navazio et al., 2007; Olah et al., 2005; Weidmann et al., 2004). In
14
soybean cell culture stably expressing the bioluminescent calcium indicator aequorin,
diffusible fungal factor(s) of small molecular mass (< 3KDa) are able to elicit AM
specific transient elevations of cytosolic free calcium (Navazio et al., 2007).
Upon contact with the fungi, the expression pattern of the PENOD11:ENOD11
changes and is restricted to the epidermal cells directly associated with visible
appressoria, flattened hyphal tips adhering to the host epidermal cells (Chabaud et al.,
2002). Studies using PENOD11:GFP-HDEL fusion in transgenic Medicago truncatula roots
confirmed that the induction of ENOD11 expression occurs in the epidermal cell after the
fungal appressorium differentiation (Genre et al., 2005). In this study, ENOD11 induction
is also synchronized with an AM specific plant cellular reorganisation occurring before
the fungus penetrates into the first cell at the epidermis or exodermis (Genre and
Bonfante, 2007; Genre et al., 2005). Indeed, from the site of the infection, a pre-
penetration apparatus (PPA) is formed. The PPA is a trans-cellular column of cytoplasm,
rich in cytoskeletal elements and endoplasmic reticulum which define the future path for
the hyphal penetration (Genre et al., 2005). The assembly of the PPA is coupled with an
initial repositioning of the nucleus at the site of fungal adhesion and subsequent
transcellular migration (Genre et al., 2008; Genre et al., 2005). Using vital lipophilic
fluorescent dye, a putative membrane structure that colocalizes with the cytoplasmic
column and associated PPA is labelled. This suggests that the PPA could be a player in
the synthesis of the membrane that surrounds and isolates the infection hypha from the
cell cytoplasm (Genre et al., 2005). Once the PPA and the perifungal membrane have
traversed the cell lumen, the fungal hypha penetration starts.
The fungus progresses through exodermal and outer cortical cells in a PPA
dependent manner and reaches the apoplast between inner cortical cells where it
proliferates (Genre et al., 2008; Parniske, 2004). This intercellular hyphal progression is
accompanied by cytoplasmic reorganisation, nuclear enlargement and repositioning
opposite to the site of fungal contact of the inner cortical cells (Genre et al., 2008). Then,
without apparent appressorium formation, the fungus invades the inner cortical cells
where it differentiates an ephemeral tree-like structure, the so-called arbuscule, which
will reach full development within several days, and then senesce (Alexander et al., 1988;
Javot et al., 2007). The arbuscule is enveloped in a membrane known as periarbuscular
membrane, derived from the host plasma membrane. The resulting surface between the
15
periarbuscular membrane and the arbuscule membrane constitutes a symbiotic interface
for nutrient exchange between the two symbionts (Gianinazzi-Pearson, 1996).
The arbuscular mycorrhizal fungi obtain carbohydrates from the host and in
exchange improve the plant acquisition in mineral nutrients, specially phosphate but also
nitrogen (Govindarajulu et al., 2005; Pearson and Jakobsen, 1993; Smith et al., 2003).
Today, few periarbuscular membrane transporters have been identified. The up-regulation
of plant proton ATPases during AM interaction as well as their specific expression in
arbuscule containing cells, suggest their involvement in the AM mechanism (Gianinazzi-
Pearson et al., 2000; Murphy et al., 1997). Furthermore, expression profiling studies have
revealed upon AM inoculation specific up-regulation of plant high-affinity nitrate
transporter as well as plant hexose transporter functionally characterized as glucose and
fructose transporter in yeast (Harrison, 1996; Hohnjec et al., 2005). So far, the most
characterized transporter involved in the AM nutrient exchange is the Medicago
truncatula phosphate transporter, MtPT4 (Harrison et al., 2002). Also mycorrhiza-
induced, MtPT4 localize to the periarbuscular membrane where it contributes to the plant
phosphate uptake (Harrison et al., 2002; Javot et al., 2007). Without MtPT4, the
arbuscules die prematurely (Javot et al., 2007). Recently the arbuscular mycorrhizal
molecule involved in the induction of MtPT4 tomato and potato orthologous has been
identified (Drissner et al., 2007). By chromatography and mass spectrometric analyses,
the active component was characterized as a lyso-phosphatidylcholine (Drissner et al.,
2007). Today, the receptor to lyso-phosphatidylcholine is unknown.
Similarly to AM symbiosis, a molecular dialogue is established between the plant
host and rhizobia in order to establish the RNS. While in the AM formation there is little
host specificity, the rhizobia-legume interaction is typically specific. The host specificity
is controlled at several levels of the molecular dialogue. At the first step of the
recognition, the bacteria perceive flavonoid, chemo-attractant, secreted by the host. The
flavonoids induce the production of a bacterial-nodulation factor, lipo-chitin-
oligosacharides, so-called Nod-factor. The nature of the flavonoids play a role in the
specific activation of the transcriptional regulator NodD which induces the expression of
the nod genes involved in the production of the Nod factor (Cardenas et al., 1995; Fisher
and Long, 1992; Long, 1996). The Nod factor is the major player of the host specificity
16
and consists of a β-1,4-linked N-acyl-D-glucosamine backbone of three to five units, N-
acetylated at the nonreducing-terminal residue by a fatty acid and containing a variety of
additional decorations at the end reducing or non-reducing terminal residue, or both
(Lerouge et al., 1990; Spaink et al., 1991). The length of the glucosamine backbone, the
modification of the terminal sugar residue or the nature of the acyl chain can all define
the biological activity and host specificity of the Nod factor (Carlson et al., 1995).
The recognition by the host plant of the Nod-factor-secreting rhizobia elicits the
growth of the root hair cell which leads to deformation and “Shepherd´s crook”-like
curling of the root hair tip within hours (Heidstra et al., 1994; Yao and Vincent, 1969).
The application of the Nod-factor alone is sufficient to induce the transient actin
fragmentation which precedes the root hair tip deformation and curling (Cardenas et al.,
1998), as well as the activation of early nodulation genes expression like ENOD11 and
cortical cells division (Catoira et al., 2001; Esseling et al., 2003; Truchet et al., 1991).
Entrapped in the curl, rhizobia enter the root hair via degradation of the cell wall,
invagination of the plasma membrane and deposition of new plant and bacterial materials
forming a new tubular structure, the infection thread (IT) (Ridge and Rolfe, 1985;
Turgeon and Bauer, 1985). The IT grows centripetally towards the cortex with rhizobia
continuously dividing (Napoli and Hubbell, 1975; Ridge and Rolfe, 1985). Similarly to
AM penetration, a reorganisation of the cytoskeleton precedes the infection thread
progression. Cytoplasmic bridge from one side of the cell to the other, the so-called pre-
infection thread (PIT), is formed which guides the infection thread passage (Timmers et
al., 1999; van Brussel et al., 1992). Likewise to the AM pre-penetration apparatus, the
PIT is closely connected to the nucleus (Timmers et al., 1999). Concomitant with the
epidermal infection thread progression, the cortical cells below the site of infection divide
to form the nodule primordium (Yang et al., 1994). The nature of the cortical cells which
differentiate into meristematic cells is determined by the plant and will specify the type of
nodule; indeterminate or determinate (Hirsch, 1992). When the infection thread reaches
the primordium, the rhizobia are released. They enter the cytoplasm via invagination of
the host-cell membrane. Similarly to the arbuscule, the bacterial surface is not in direct
contact with the cytoplasm but remains surrounded by a plant-derived membrane, the
peribacteroid membrane, and together form a so-called symbiosome (Oke and Long,
1999). In this microaerobic environment, the bacteria differentiate into bacteroids in
17
which nitrogen reduction into ammonia occurs (Oke and Long, 1999). On the
peribacteroid membrane that is highly permeable to NH4+, ammonium transporter has
been characterized (Kaiser et al., 1998; Niemietz and Tyerman, 2000).
Among legumes, two types of root nodules are found; determinate and
indeterminate nodules (Hirsch, 1992). On pea and alfalfa roots for example,
indeterminate nodules are formed. The cell division that initiates this type of nodule takes
place in the inner cortex and leads to the formation of a nodule with a persistent apical
meristem (Robertson et al., 1985). The constant addition of new cells leads to the
formation of an egg-shape nodule. In comparison, in lotus and soybean roots, determinate
nodules are developed from outer cortical cells. The meristematic activity stop in an early
stage and the subsequent nodule growth depends on cell expansion leading to a mature
spherical nodule (Stougaard, 2000). In most species, nodules are visible within 7 days
after inoculation (Albrecht et al., 1999).
1.2 Root-nodule-symbiosis early ion fluxes
Using a combination of electrophysiology and fluorescence microscopy with ion
sensitive dyes, specific ion fluxes preceding root hair deformation have been measured in
response to Nod factor (Felle et al., 1998; Shaw and Long, 2003).
Within one minute after addition of at least 10 nM Nod factor, a rapid calcium
influx into the cytoplasm of the root hair tip of alfalfa is recorded (Ehrhardt et al., 1996;
Felle et al., 1998). The calcium influx can raise the intracellular calcium concentration to
1500 nM at the root hair tip (Cardenas et al., 1999). In Medicago truncatula the calcium
influx is biphasic, with a first increase in calcium followed by an elevated level of
calcium lasting for several minutes (Shaw and Long, 2003). The calcium influx induces
secondary responses including membrane depolarization of 10-15 mV and transient
intracellular alkalinization of 0,3 pH units (Ehrhardt et al., 1992; Felle et al., 1995; Felle
et al., 1996; Felle et al., 1998; Kurdjian, 1995). By using stationary ion-selective
extracellular electrodes, a chloride efflux triggered by the calcium influx is proved to be
responsible for the membrane depolarization (Felle et al., 1998). The charge balance
which eventually stops the depolarization and initiates the repolarization is provided by
18
potassium efflux (Felle et al., 1998). During Nod-factor-induced membrane
depolarization, addition of H+-ATPase inhibitor abolishes the membrane repolarization
suggesting that a proton efflux is responsible for the membrane potential recovery (Felle
et al., 1998). Concomitant to the membrane depolarization, the calcium influx triggers a
transient alkalinization of the root hair surface. Upon H+-ATPase inhibitor treatment, the
alkalinization becomes persistent suggesting that it could be due to a transient Nod factor-
induced inhibition of a H+-ATPase (Felle et al., 1998). So far, no causal link has been
proved between the membrane depolarization and the transient alkalinization.
Subsequently, with a delay of 10-20 min, transient oscillations of the calcium
concentration rising from 50 to 600 nM, so-called calcium spiking, are observed around
the nucleus (Ehrhardt et al., 1996; Shaw and Long, 2003). Due to the nucleoplasmic and
perinuclear localization of the Ca2+ spiking, cisterns of the endoplasmic reticulum (ER)
and the nuclear envelope are likely to be the corresponding Ca2+ stores (Oldroyd and
Downie, 2006). The calcium oscillations, which can last 1 to 3 hours, are regular with a
mean period of 60 seconds for a single spike and can be induced with a Nod factor
concentration as low as 1 pM (Ehrhardt et al., 1996; Shaw and Long, 2003). The spike is
imposed on a baseline with a raising phase of each peak typically faster than the falling
phase (Ehrhardt et al., 1996).
So far, the calcium influx and the calcium spiking have been reported from six
and five different legumes, respectively (Cardenas et al., 1998; de Ruijter et al., 1998;
Harris et al., 2003; Miwa et al., 2006; Shaw and Long, 2003; Wais et al., 2000; Walker et
al., 2000). In addition to those reports, Nod factor fails to induce calcium spiking and
calcium influx in root hair cells of non-leguminous plants (Ehrhardt et al., 1996). All
together these results suggest that the calcium fluxes are specific and common features of
Legume-rhizobia interactions.
The calcium spiking and the calcium influx responses are spatially distinct and
appear to be causally unlinked (Shaw and Long, 2003). Various non-nodulating mutants
impaired for the calcium spiking still show calcium influx (Miwa et al., 2006; Shaw and
Long, 2003). In wild-type legumes, Nod-factor-like molecules, N-acetylglucosamine
tetramers or partially decorated Nod factors can trigger calcium spiking but fail to induce
calcium influx (Ardourel et al., 1994; Shaw and Long, 2003; Walker et al., 2000). In
calcium-spiking-deficient mutants, these molecules cannot cause the calcium spiking
19
which suggests that both stringent and non-stringent perceptions of Nod factor involve
the same Nod factor pathway (Oldroyd et al., 2001). Furthermore, the minimal
concentration of Nod factor required to induce the calcium spiking is three orders of
magnitude lower than the concentration needed to trigger the calcium influx (Shaw and
Long, 2003). Collectively, these results suggest that there is a lower stringency of Nod
factor perception required for induction of calcium spiking, whereas a relatively high
concentration and specific Nod factor structures are required to induce distinctly the
calcium influx.
Forward genetics approaches, using ethyl methane-sulfonate (EMS) or T-DNA
insertion as mutagen tools, yielded at least seven distinct mutants impaired in the
symbiotic process upstream of the calcium spiking (Miwa et al., 2006; Perry et al., 2003;
Schauser et al., 1998). Among those loci, only two, NFR1 and NFR5, are RNS specific,
whereas the five other loci are commonly involved in AM and RNS symbioses, and so-
called common SYM genes (Kistner and Parniske, 2002). NFR1 and NFR5 are required
for the calcium influx, membrane depolarisation and transient alkalinisation, as well as
noduline genes expression (Madsen et al., 2003; Miwa et al., 2006; Radutoiu et al., 2003).
The nfr1 and nfr5 mutants show no root hair deformation upon Mesorhizobium loti
inoculation (Radutoiu et al., 2003). The corresponding LysM receptor-like kinases, NFR1
and NFR5, have been shown to determine the Nod factor specificity of legume/rhizobial
symbiosis. The transfer of L. japonicus NFR1 and NFR5 to M. truncatula enables indeed
nodulation of the transformants by the L. japonicus symbiont M. loti (Radutoiu et al.,
2007). The LysM-containing extracellular domains have also been proved to be directly
involved in the Nod factor perception (Radutoiu et al., 2007). Although a direct Nod
factor binding has not been demonstrated yet, the results suggest that NFR1 and NFR5
form the Nod factor receptor complex required for the first recognition step (Radutoiu et
al., 2003; Radutoiu et al., 2007).
In all five other common symbiosis mutants impaired in the calcium spiking, so-
called symrk, nup85, nup133, castor and pollux (Table 1), the root hair deformation
phenotype is accentuated in comparison to the wild type upon incubation with rhizobia or
Nod factor (Bonfante et al., 2000; Kanamori et al., 2006; Miwa et al., 2006; Stracke et al.,
20
2002). However, no curling of the root hair tips is observed. This phenotypic
characterization suggests that the Nod factor signalling pathway is blocked downstream
of Nod factor perception and that the calcium spiking would be required for root hair
curling followed by bacterial entrapment. In these mutants, upon treatment with a high
Nod factor concentration, calcium influx is induced (Miwa et al., 2006). Considering the
amount of Nod factor required to elicit calcium influx, it is proposed that in physiological
condition calcium influx is induced when the bacteria are entrapped prior to the infection
thread formation (Miwa et al., 2006). This calcium influx would be necessary to the
bacterial entry process through infection thread formation. Support for this model comes
from the analysis of infection thread initiation in vetch by nodulation mutants of
Rhizobium leguminosarum (Walker and Downie, 2000). The expression of the R.
leguminosarum nodO gene in Rhizobium mutant can compensate the loss of the
appropriate Nod factor production and induce infection thread formation (Walker and
Downie, 2000). The nodO gene of R. leguminosarum encode for a secreted protein that
can form cation-selective channels in lipid bilayers (Sutton et al., 1994). It has been
proposed that NodO may stimulate ion flux such as calcium influx across the host plasma
membrane which will be sufficient to induce infection thread formation (Walker and
Downie, 2000). However, the infection threads formed have an irregular shape and are
arrested prematurely (Walker and Downie, 2000). This observation suggests that a
structurally specific Nod factor is necessary to pass a third “checkpoint” that controls the
infection thread progression.
In this model, at least three receptor complexes, named perception, entry and
progression receptor complexes, are required for the rhizobia to enter and progress into
the host cells. If the putative NFR1/NFR5 receptor-like kinases complex is likely to
monitor the first recognition step, other receptors and/or a different combination of
receptors may be involved at the subsequent steps. SYMRK is encoded for an active
receptor-like kinase involved downstream of NFR1 and NFR5; after treatment with
purified Nod factor, double mutants SYMRK/NRF1 or SYMRK/NFR5 respond similarly to
NFR1 or NFR5 mutants, respectively (Radutoiu et al., 2003; Stracke et al., 2002; Yoshida
and Parniske, 2005). The phenotypic results suggest that SYMRK is involved in a
signalling pathway leading to calcium spiking and curling. However, an additional role of
SYMRK in the bacterial entry process cannot be excluded. Finally, several L. japonicus
21
mutants have been described as defective for infection thread growth, including alb1,
sym7, sym8, sym10, sym80, crinkle, itd1, itd3 and itd4 (Lombardo et al., 2006; Schauser
et al., 1998; Tansengco et al., 2003; Yano et al., 2006). For some of those mutants, the
bacteria stay entrapped in the infection pocket, while in other mutants abnormal infection
threads are formed. The identification of these different genes will give new insight into
the mechanism of the entry and of the infection thread growth “checkpoints”.
Table 1.
Common L. japonicus SYM genes required for AM and RNS. L. japonicus genes Characteristics Orthologous
M. truncatula P.sativum Reference(s)
SYMRK (SYM2) a
Hac-, no Ca2+ spiking DMI2 PsSYM19 (Endre et al., 2002; Stracke et al., 2002)
POLLUX (SYM23 and SYM86) a
Hac-, no Ca2+ spiking (Kistner et al., 2005)
CASTOR (SYM4 and SYM71) a
Hac-, no Ca2+ spiking
DMI1* PsSYM8*
(Bonfante et al., 2000; Ovtsyna et al., 2005; Zhu et al., 2006)
NUP85 (SYM24, SYM73 and SYM85) a
Hac-, no Ca2+ spiking - - (Saito et al., 2007)
NUP133 (SYM3 and SYM45) a
Hac-, no Ca2+ spiking - - (Kanamori et al., 2006)
CCaMK (SYM15 and SYM72) a
Hac-, Ca2+ spiking DMI3* PsSYM9*/ PsSYM30*
(Levy et al., 2004; Mitra et al., 2004; Ovtsyna et al., 2005; Tirichine et al., 2006)
CYCLOPS (SYM82, SYM6 and SYM30) a
Iti-, Ca2+ spiking IPD3* PsSYM36* (Messinese et al., 2007; Tsyganov et al., 2002; Yano et al., 2006)
* putative orthologous of L. japonicus gene; a previous nomination used in the studies referenced; Hac-, no root hair curling ; Iti-, blockage at the stage of infection thread initiation.
22
1.3 Dissection of the calcium-spiking pathway
Both the rhizobial bacteria and the mycorrhizal fungi trigger nuclear-localized calcium
spiking via the common symbiotic signalling pathway, which includes at least seven
proteins (Table 1). Although components of the same signalling pathway are involved,
the fungal and bacterial calcium signatures generated are very different in terms of
frequency and amplitude (Kosuta et al., 2008). The AM-induced calcium spiking has an
irregular spiking frequency which produces a 74-113 nM calcium change which is only
17 % of Nod factor-induced calcium change (Kosuta et al., 2008). Furthermore, in
contrast to the Nod-factor induced calcium spiking which is repetitive and periodic, the
AM-induced calcium spikes differ with each repetition (Kosuta et al., 2008). How those
two different calcium signatures are generated and decoded by a signalling pathway
involving identical components is still unclear. Among the common symbiotic proteins
acting downstream of the calcium spikes, a calcium/calmodulin-dependent protein kinase,
CCaMK, has been identified and constitutes an obvious candidate involved in decoding
the calcium signatures (Gleason et al., 2006; Godfroy et al., 2006; Levy et al., 2004;
Mitra et al., 2004; Tirichine et al., 2006).
Today, no calcium channels responsible for the generation of nuclear localized-
calcium spiking have been identified in plants. In animal cells, Ca2+-permeable channels
such as ryanodine receptors and inositol triphosphate receptors (InsP3-R) localized in the
nuclear envelope or ER are involved in the generation of Ca2+ oscillation around the
nucleus (Furuichi et al., 1989; Takeshima et al., 1989). These channels can be activated
by secondary messengers such as nicotinic acid adenine dinucleotide phosphate
(NAADP), 1-(5-phospho-b-D-ribosyl) adenosine 5-phosphate cyclic anhydride (cADP-
Rib), inositol-1,4,5-triphosphate (InsP3) and Ca2+ (Gerasimenko and Gerasimenko, 2004).
InsP3 is generated from degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by a
phosphoinositide-dependent phospholipase C (PI-PLC). The mammalian PI-PLC is
primarily activated via heterotrimeric G-proteins (Fukami, 2002; Ross and Higashijima,
1994). Mastoparan, an agonist of heterotrimeric G-proteins in animal cells, activates PI-
PLC and induces Ca2+ fluxes (Ross and Higashijima, 1994). Although the precise mode
of action of mastoparan in plant cells is unclear (Miles et al., 2004; Pingret et al., 1998), it
triggers Ca2+ spiking (Pingret et al., 1998; Sun et al., 2007), as well as nodulation gene
23
expression in Medicago truncatula (Pingret et al., 1998; Sun et al., 2007). In vetch roots,
a synthetic analogue to mastoparan, mastoparan-7, elicits root hair deformation and
activates both PI-PLC and phospholipase D (PLD), which results in an accumulation of
phosphatidic acid (PA) and diacylglycerol pyrophosphate (DGPP), similarly to Nod
factor treatment (den Hartog et al., 2001). Furthermore, the application of PI-PLC and
PLD antagonists inhibits Nod factor-induced root hair deformation, PA accumulation and
expression of nodulation genes (Charron et al., 2004; den Hartog et al., 2001; den Hartog
et al., 2003; Pingret et al., 1998). The PI-PLC antagonist, U-73122, also inhibits Nod
factor-elicited calcium spiking (Charron et al., 2004; Engstrom et al., 2002). All together,
these results suggest that similar signalling transduction pathways lead to Ca2+
oscillations in plants and animals.
However, ryanodine receptors or InsP3-R have not been identified in the
completely sequenced Oryza sativa and Arabidopsis thaliana genomes, suggesting
alternative, yet unidentified channels are involved (Nagata et al., 2004). Despite the
absence of clear homologs in plants, both NAADP and cADP-rib elicit Ca2+ release from
plant ER-derived vesicles (Navazio et al., 2000; Navazio et al., 2001). Furthermore, an
inhibitor of InsP3-activated channels, 2-aminoethoxydiphenylborate, blocks the Nod
factor induced Ca2+ spiking (Engstrom et al., 2002). Collectively, these results suggest
the presence of yet-to-be identified plant Ca2+ channels activated similarly to the Ca2+
channels responsible for Ca2+ oscillations in animal cells.
1.4 Castor and pollux mutants and aim of the study
The absence of Ca2+ spiking, root hair curling, infection thread and cortical cell division
in castor and pollux mutants, suggests that CASTOR and POLLUX act early in a signal
transduction chain leading to the activation of calcium-spiking and bacterial entrapment
in curled root hair. It is therefore postulated that CASTOR and POLLUX are necessary to
allow the passage of the bacteria through the epidermis layer (Bonfante et al., 2000;
Kistner et al., 2005; Miwa et al., 2006; Schauser et al., 1998).
Similarly, fungal infection attempts on castor and pollux mutants generally abort
within the epidermis (Bonfante et al., 2000; Kistner et al., 2005). The AM fungi develops
24
appressoria from which hyphae grows between two epidermal cells, before the
progression aborts in the extracellular space. There, hyphae often show balloon-like
swellings or other deformation (Bonfante et al., 2000; Kistner et al., 2005). Interestingly,
two alleles of castor, castor-1 and castor-2, present different AM phenotypic strength
(Novero et al., 2002). In roots of castor-2 mutant, fungal penetration beyond the
epidermis after appressoria formation is never observed. In comparison, in roots of
castor-1, fungal hyphae are mainly blocked at the intercellular-space of the cortex before
arbuscule formation. In rare cases, arbuscules are formed. These observations suggest
that castor could be required for infection of both epidermal and cortical cells by AM
fungi (Bonfante et al., 2000; Novero et al., 2002).
The CASTOR and POLLUX genes were isolated through positional cloning and by
their mutual homology. Using the collection of microsatellite (SSR) markers available for
L. japonicus (Hayashi et al., 2001), the CASTOR and POLLUX genes were positioned
close to the bottom ends of chromosome 1 and chromosome 6, respectively. Five castor
alleles were independently mapped to the south end of chromosome 1, close to SSR
marker TM0105 (Nakamura et al., 2002). Starting with this marker, a BAC/TAC
(Kawasaki and Murakami, 2000; Nakamura et al., 2002) contig was constructed which
extended into the telomeric region (Figure 2). Genetic mapping was affected by a 145 kb
long inversion between the parental lines B-129 and MG-20 (Kawaguchi et al., 2001)
with no recombination events observed upon inspection of 1,833 individuals of five
different F2 populations, delimiting the CASTOR locus to a region of about 240 kb.
PCR-marker TB2R could not be amplified from individuals homozygous for the G00472
or G00716 alleles. PCR and southern blot analysis indicated that both alleles have large
deletions of more than 20 kb encompassing the CASTOR gene, and subsequent sequence
analysis revealed that marker TB2R is located in a CASTOR intron.
The POLLUX gene was found to co-segregate with SSR marker TM0885. A TAC
clone carrying SSR TM0885 was completely sequenced, and a candidate gene with high
homology to CASTOR was identified (Figure 1).
25
Figure 1. Positional cloning of CASTOR and POLLUX genes (Imaizumi-Anraku et al., 2005). (A) Genetic and physical map around the CASTOR locus. Positions of flanking markers are indicated together with the number of recombinant plants/total number of mapping individuals. Designations of large insert clones originating from parental ecotype B-129 are; G1, BAC156-1E; G2, BAC124-7B; G3, BAC324-1D; G4, BAC104-3F; G5, BAC33-3E; and from parental ecotype MG-20: M1, LjT17M09; M2, LjT62O06; M3, LjT02K14; M4, LjT45I15; M5, LjT20F11 and M6, LjT46G19. An inverted region of 145kb between the genomes of B-129 and MG-20 was detected, which is probably responsible for the observed suppression of recombination around the CASTOR locus (0 cM). BAC end markers within the inverted region are: open oval, TB21R; closed oval, TA3F; open rectangle, TB2R. The TB7R marker located in the north side delimits CASTOR to ~240kb. (B) Candidate genes predicted within the target region. LRRPK, leucine-rich repeat receptor kinase; 26SP, 26S proteasome regulatory subunit 7; RE, retro-element; EP, Avr9/Cf-9 elicited protein; PUM, pumilio-family RNA-binding protein; RHZF, Ring H2 zinc finger protein; PME, pectin methyl esterase; IF2, initiation factor 2 subunit; OXI, oxido-reductase; PP, polyprotein; MYB, myb family protein; GAG, gag-pol polyprotein; FIP; VirF-interacting protein FIP1; ANK, ankyrin-like protein; REV; reverse transcriptase; unlabelled, hypothetical protein. (C) Genetic and physical map of the POLLUX locus. Positions of the flanking markers and the number of recombinant plants in the mapping populations are indicated. Abbreviations G6, G7, M7, M8 and M9 refer to large insert clones BAC131-3b, BAC45-6C, LjT39N07, LjB22b22 and LjT45B09, respectively. (D) Genes predicted on the cosegregating TAC clone LjT45B09; WD40, WD40 repeat protein; NifU, putatively involved in Fe-S cluster synthesis; PPR, pentatricopeptide repeat protein; DNABP, DNA binding protein; LRRPK, leucine-rich repeat receptor kinase; unlabelled, hypothetical protein.
26
In the present study, we aim to characterize CASTOR and POLLUX at molecular
levels to unravel their role in the early common symbiosis-signalling pathway.
Figure 2. A model for the early symbiosis signalling pathway in the epidermis. The receptor-like kinases, NFR1 and NFR5, are required for the Nod-factor perception, which leads to the generation of the Nod factor-induced calcium spiking via at least five common symbiotic components; SYMRK, CASTOR, POLLUX, NUP85 and NUP133. Although no receptor has been cloned, an equivalent receptor-like kinase is presumed to exist for the recognition of mycorrhizal factor which lead to the mycorrhiza-induced calcium spiking via the same common symbiotic pathway (Kosuta et al., 2008). The calcium spiking is localized around the nucleus. The calcium storage compartment is believed to be the cisterns of the ER and the nuclear envelope. A yet-to-be identified calcium channel is responsible for the calcium release. The potassium influx triggers by the nuclear-localized cation channels, CASTOR and POLLUX, might compensate for the resulting charge imbalance created by the calcium released. Calcium ATPases are believed to be involved in the replenishment of the calcium storage. The CCaMK positioned downstream of the calcium spiking is believed to be involved in deciphering the calcium traces leading to either nodulation or mycorrhization programming. PM, Plasma membrane; ONM, Outer nuclear membrane; INM, Inner nuclear membrane.
We demonstrate that CASTOR and POLLUX can form two distinct
homocomplexes. In electrophysiological assays or yeast complementation experiments,
we show that both complexes are cation channels permeable to potassium. We further
proved that CASTOR is localized in the nuclear envelope of L. japonicus root cells.
Therefore, we propose a model in which CASTOR and POLLUX act as counter ion
channels that mediate compensation of the charge imbalance associated with the calcium
SYMRK NFR1 NFR5
NodF Mycorrhiza Factor(s)
Ca2+
CCaMK
Ca2+channel
PM
Cytoplasm
Nucleus
Ca2+ ATP
ATP
CASTOR
POLLUX
K+ K+
K+
K+
Signal transduction
Channel activation
Intracellular infection
NUP133 NUP85
ONM INM
Nodule organogenesis
27
efflux. Alternatively, and not mutually exclusively, they may modulate the nuclear
membrane potential leading to the activation of calcium channels responsible for calcium
spiking (Figure 2).
28
2. Results
2.1 CASTOR and POLLUX identification
Sequencing of genomic DNA in the CASTOR and POLLUX targets interval identified
non-silent mutations within nine castor mutants and ten pollux mutants, respectively.
Additional mutants were identified through Targeted Induced Local Lesions in Genomes
(TILLING) (Perry et al., 2003). Twenty-seven and nineteen additional castor and pollux
mutants, respectively, were identified through TILLING and sequencing of candidate
genes (Table 2 & 3).
Full-length complementary DNAs (cDNA) sequences corresponding to the
CASTOR and POLLUX genes were obtained (Imaizumi-Anraku et al., 2005). Both genes
encompass 12 exons and are subject to alternative splicing, as discovered through
sequence analysis of cDNA clones (Figure 3). In total, three CASTOR and two POLLUX
alternative spliced (AS) variants were amplified (Figure 3).
Figure 3. Alternative-spliced variants of CASTOR (A) and POLLUX (B). Exons are indicated by upper gray boxes and introns by lower triangles. Alternative-spliced products have been found with the following change; red arrow head: alternative acceptor sites, empty red arrow head: alternative donor sites, red triangle: intron retention. Three CASTOR alternative-spliced variants were identified: 4-nt insertion at the 3´end of the 1st exon, 9-nt insertion at the 5´end of the 2nd exon, retention of the 7th exon leading to a premature stop codon. Two POLLUX alternative-spliced variants were identified: 16-nt deletion at the beginning of the 10th exon, and retention of the first 116-nt of intron 10 leading to a premature stop codon. Blue arrow head: position of the mutation in the indicated mutant allele.
1kb
castor-12 G271A/W93STOP
A castor-1
Del L480-A481
pollux-5 G1207A/W321STOP
B
29
Table 2.
List of castor mutant alleles Allele Progenitor/
mutant line Genomic mutationa
Effect Nodulation phenotypeb
AM phenotypeb
Detected by
TILLING
Geno-type of
M2 plantc
Refe- rences
castor-1 282-227 A2776-C2781 deletion
L479-A480 - - This study
castor-2 EMS1749 G1057 to A A264 to T - - NODPOP HOM This study
castor-3 EMS46 G1655 to A G383 to E - - NODPOP HOM This study
castor-4 25-5A C778del Frame shift - n.d. (Imaizumi-Anraku et al., 2005)
castor-5 24-8B/SL1937-2 G3053 to A W483 to stop -,- n.d.,- (Imaizumi-Anraku et al., 2005)
castor-6 N5 C9686 to T P698 to L - n.d. (Imaizumi-Anraku et al., 2005)
castor-7 N10 G8623 to A V598 to I - n.d. (Imaizumi-Anraku et al., 2005)
castor-8 G00472 > 20 kb deletion CASTOR deletion
- n.d. (Imaizumi-Anraku et al., 2005)
castor-9 G00716 > 20 kb deletion CASTOR deletion
- n.d. (Imaizumi-Anraku et al., 2005)
castor-10 G00862 117 bp deletion from exon 4 to intron 4
frame shift - n.d. (Imaizumi-Anraku et al., 2005)
castor-11 M89-27 T10371 del. frame shift - n.d. (Imaizumi-Anraku et al., 2005)
castor-12 SL3251-2 G279 to A W93 to stop - - NODPOP HOM This study
castor-13 SL0820d-2,3 G2672 to A D444 to N (-*),(-) (n.d.), (-) NODPOP, NODPOP
HET, HOM
This study
castor-14 SL1715-2,3,4,5,6 G9871 to A A760 to T -*,-,-*,-*,-* n.d., -, n.d., n.d., n.d.
all in NODPOP
all HOM This study
castor-15 SL1966-3 C1013 to T T249 to I - - NODPOP HOM This study
castor-16 SL3160-3 G1655 to A G383 to E - - NODPOP HOM This study
castor-17 SL6812-2 G3738 to A R590 to H +/- - NODPOP HOM This study
castor-18 KL549 n.d. n.d. - n.d. (Sandal et al., 2006)
castor-19 G00532-21 > 20 kb deletion CASTOR deletion
- n.d. (Sandal et al., 2006)
castor-20 LKL186-4 C9827 to G S745 to stop - n.d. NODPOP HOM (Sandal et al., 2006)
castor-21 B32-BAe G1594 to A E363 to K (-) (+) (Murray et al., 2006)
castor-22 S41-1, SL1937-2 G3053 to A W483 to stop (-),- (+),- (Murray et al., 2006)
castor-23 B5-2e G1655 to A G383 to E (-) (-) (Murray et al., 2006)
castor-24 B68-Be G10353 to A W840 to stop (-) (-) NODPOP HET (Murray et al., 2006)
castor-25 S46-1e,f G1251 to A W328 to stop (-) (-) NODPOP HOM (Murray et al., 2006)
castor-26 SL6908-2 G1088 to A G274 to D - - NODPOP HOM (Perry et al.,i)
castor-27 SL5136-3 G1503 to A splice site - - NODPOP HOM (Perry et al.,i) castor-28 282-643M1M1M3g A3214 to G D537 to G n.d. n.d. NODPOP HOM (Perry et al.,i) castor-29 SL5210-1 G9105 to A E672 to K -* n.d. NODPOP HET (Perry et al.,i) castor-30 SL1415-2,3,4 G9785 to A S731 to N -,-*,-* -,n.d.,n.d. all in
NODPOP all HET (Perry et al.,i)
castor-31 SL4162-2h/ SL5768-3
C172 to G R58 to G (-), +/-* (-),n.d. (Perry et al.,i)
castor-32 SL6453-2 T960 to A V231 to V - n.d. (Perry et al.,i) castor-33 SL1371-2 G3481 to A intron 7 - n.d. NODPOP HET (Perry et al.,i) castor-34 SL6077-2 G1915 to A intron 4 -* n.d. NODPOP HOM (Perry et al.,i) castor-35 SL1052-1 G468 to A E122 to K n.d n.d. NODPOP HET (Perry et al.,i) castor-36 SL0818-1 C3354 to T intron 7 n.d. n.d. NODPOP HET (Perry et al.,i)
aThe CASTOR genomic sequence is deposited to GenBank under accession number AB162016 and is used as Lotus japonicus Gifu reference. Adenine of the ATG initiator codon is designated as position 1.bPhenotypes that are scored only in the initial M2 generation are denoted by *; no star stands for additional screening in the M3 generation. In the nodulation phenotype “+/-” designates non-functional symbiosis (e.g. few small white nodules). Annotation in brackets indicate lines harbouring more than one mutation in nodualtion genes. cGenotype of progenitor or mutant plant in the M2 generation is given as HOM= homozygous or HET=heterozygous.dLines SL0820-2,3 carry the mutant alleles castor-13 and ccamk-8. eLines contain in addition a har1-1 mutation. fLine S46-1 carries the mutant alleles castor-25, nin-10, and har1-1. gLine 282-643M1M1M3 carries the mutant alleles castor-28, ccamk-8, and nup85-9. hLine SL4162-2 carries the mutant alleles castor-32 and pollux.i Manuscript in preparation.
30
Table 3. List of pollux mutant alleles
Allele Progenitor/ mutant line
Genomic mutationa
Effect Nodulation phenotypeb
AM phenotypeb
Detected by TILLING
Geno-type of
M2 plantc
Re-ference
pollux-1 EMS70 G1152 to A G303 to S - - NODPOP HOM This study
pollux-2 EMS167 G1152 to A G303 to S - - NODPOP HOM This study
pollux-3 29-2A T5668 to A L841 to stop - n.d. This study
pollux-4 SL0571-2 C434 to T S145 to F - - NODPOP HET This study
pollux-5 SL3130-2,4 G1204 to A W320 to stop -*,- n.d.,- both in NODPOP
both HOM This study
pollux-6 SL5691-3,4 C1210 to T S322 to F +*,-* n.d., n.d. both in NODPOP
both HOM This study
pollux-7 SL1899-2,4 G2313 to A G530 to E +/-,+ n.d., n.d. both in NODPOP
both HOM This study
pollux-8 SL0159-2,3,5,6 G5486 to A W780 to stop -,-*,-,- n.d.,n.d.,-,- all in NODPOP
all HOM This study
pollux-9 SL0405-2,3,5,6 G5634 to A E830 to K -*,-,-*,-* n.d.,n.d.,n.d.,n.d.
all in NODPOP
all HOM This study
pollux-10 SL1070-2 G6021 to A G882 to S - n.d. NODPOP HOM This study
pollux-11 B12-1Ae G1205 to A W320 to stop (-) (-) (Murray et al., 2006)
pollux-12 B50-Ce G1644 to A splice site (+/-) (-) NODPOP HOM (Murray et al., 2006)
pollux-13 S49-De G5682 to A splice site (-) (-) NODPOP HOM (Murray et al., 2006)
pollux-14 Sup3e C4804 to T P719 to L (-) (-) NODPOP HOM (Murray et al., 2006)
pollux-15 S24-1Bg A458-C463 del Q153-H154 del n.d. (+) NODPOP HOM (Murray et al., 2006)
pollux-16 SL5998-2,3,4,5,6 G496 to A splice site -,-*,-*,-*,-* n.d.,-, n.d., n.d., n.d.
all in NODPOP
all HOM (Perry et al.,m) pollux-17 SL1657-2 G1147 to A G301 to E - - NODPOP HOM (Perry et al.,m) pollux-18 SL0317-3h G1179 to A G312 to S (+*) n.d. NODPOP HET (Perry et al.,m) pollux-19 SL5821-3,5 G1644 to A splice site -,-* n.d.,- both in
NODPOP both HOM (Perry et al.,m)
pollux-20 SL0729-6,10 G3388 to A W651 to stop -*,-* -,- both in NODPOP
both HOM (Perry et al.,m) pollux-21 SL1075-2 G3416 to A splice site - - NODPOP HOM (Perry et al.,m) pollux-22 SL0283-4 G4698 to A E684 to K n.d. n.d. NODPOP HET (Perry et al.,m) pollux-23 SL4162-2i C341 to T T114 to I (-) (-) NODPOP HOM (Perry et al.,m) pollux-24 SL1913-7,8k C1922 to T intron 4 (+),-* (+), n.d. both in
NODPOP both HET (Perry et al.,m)
pollux-25 SL5546-2,3 C3568 to T intron 8 +*,+* n.d. both in NODPOP
both HET (Perry et al.,m) pollux-26 SL0405-4 G2837 to A intron 6 -* n.d. NODPOP HET (Perry et al.,m) pollux-27 SL0299-2 G2672 to A intron 6 -* n.d. NODPOP HOM (Perry et al.,m) pollux-28 SL0456-2l T5897 to G intron 11 (+) n.d. NODPOP HET (Perry et al.,m) pollux-29 SL0387-3 G4646 to A L666 to L -* n.d. NODPOP HOM (Perry et al.,m)
aThe POLLUX genomic sequence is deposited to GenBank under accession number AB162017 and is used as Lotus japonicus Gifu reference. Adenine of the ATG initiator codon is designated as position 1. bPhenotypes that are scored only in the initial M2 generation are denoted by *; no star stands for additional screening in the M3 generation. In the nodulation phenotype “+/-” designates non-functional symbiosis (e.g. few small white nodules). Annotation in brackets indicate lines harbouring more than one mutation in nodualtion genes. cGenotype of progenitor or mutant plant in the M2 generation is given as HOM= homozygous or HET=heterozygous. dpollux-1 and pollux-2 are the same allele eLines contain in addition a har1-1 mutation fpollux-12 and pollux-19 are the same gLine S24-1B carries the mutant alleles pollux-15 and symrk-28 hLine SL0317-3 carries the mutant alleles nup133-14 and pollux-18 iLine SL4162-2 carries the mutant alleles castor-32 and pollux-23 kLine SL1913-7 carries the mutant alleles pollux-24 and symrk-60 lLine SL0456-2 carries the mutant alleles nfr1-5 and pollux-28 m Manuscript in preparation
31
To ascertain the role of these AS in the symbiosis process, we tested whether CASTOR
and POLLUX cDNA under the regulation of the cauliflower mosaic virus 35S promoter
(P35S) can mediate nodulation and arbuscule formation in castor-12 and pollux-5
mutants, respectively (Table 2, Table 3 and Figure 3). In both castor-12 and pollux-5
mutants, a guanosine residue is mutated to an adenosine leading to premature termination
codons (PTCs) at the positions W93 and W321, respectively. The PTCs are produced
before any alternative splicing sites, depleting the mutant of AS variants (Figure 3).
Transgenic roots of the castor-12 and pollux-5 plants carrying CASTOR and POLLUX
full-length cDNA, respectively, formed pink nodules after 5 weeks inoculation with M.
loti (Table 4). Castor-12 and pollux-5 are also impaired in AM. CASTOR and POLLUX
full-length cDNA restored the AM defect in transgenic roots of this line (Table 4). All
together, the results confirm the identification of CASTOR and POLLUX and demonstrate
that CASTOR and POLLUX full-length cDNA are sufficient to support both fungal and
bacterial endosymbiosis in L. japonicus without AS variants.
Table 4.
Restoration of root nodules symbioses in L. japonicus castor-12 and pollux-5 mutants
Plant genotype Transgene Nod + Nodule/Nodulated plant
AM +
L. japonicus wild-type
marker only* 30/30 13.0 15/15
pollux-5 POLLUX 45/45 12.2 8/8 pollux-5 marker only* 0/30 0 0/6 castor-12 CASTOR 24/24 11.4 10/10 castor-12 marker only* 0/18 0 0/10
Numbers refer to A. Rhizogenes root systems of L. japonicus mutants or wild-type. Nod +, number of root systems showing nodules containing bacteria; AM +, number of roots system showing AM 2 weeks after inoculation with Glomus intraradices BEG 195 on nodulated plants; *Plants transformed with the binary vector pH7WG2D (Karimi et al., 2002) lacking CASTOR and POLLUX expression cassettes.
2.2 Sequence analyses
CASTOR and POLLUX are two homologous genes which share 62.6% identity. Southern
blot experiment indicates that CASTOR – POLLUX homologues exist in one or two
copies in other dicotyledonous and monocotyledonous plants including Medicago
truncatula, Pisum sativum, Glycin max, Arabidopsis thaliana and Oryza sativa
32
(Imaizumi-Anraku et al., 2005). BLASTN searches of National Center for Biotechnology
Information (NCBI) identified expressed sequence tags (ESTs) in the same species for
both CASTOR and POLLUX, confirming that the sequence homologues represent
expressed genes. BLASTP searches of NCBI non-redundant protein sequences databases
predicted homologs in the same monocotyledonous and dicotyledonous species as well as
more distant homologs in the moss Physcomitrella patens. CASTOR- and POLLUX-
related sequences were not identified in the full sequence genomes of animals, fungi, or
green algae. However, relative close homologs were identified in bacteria genera, namely
Nocardiodes sp. JS61, Mesorhizobium loti and Streptomyces ambofaciens.
Figure 4. Unrooted radial phylogenetic tree of CASTOR and POLLUX homologs. Homologous full-length protein sequences with E value ≤ 10-41 were aligned using ClustalX and MacClade programs. The protein tree was constructed using the consensus parsimony method (PHYLIP 3.67 program). Percentage bootstrap support based on 1000 replicates is given to the side of each branch. L.j., Lotus japonicus; M.t., Medicago truncatula; A.t., Arabidopsis thaliana; O.s., Oryza sativa; V.v., Vitis vinifera; P.p., Physcomitrella patens; S.a., Streptomyces ambofaciens; N.sp., Nicardioides sp.; M.l., Mesorhizobium loti; P.s., Pisum sativum. L.j. CASTOR, gi:62286596; L.j. POLLUX, gi:62287141; O.s.1, gi:115441303; O.s.2, gi:115456529, O.s.3, gi:125546404; M.t.DMI1, gi:62286545; P.s.SYM8, gi:161105392; V.v.1, gi:157353778; V.v.2, gi:157354359; A.t.1, gi:186530826; A.t.2, gi:186519621; A.t.3, gi:30679833; P.p., gi:168003777; S.a., gi:117165168; N.sp., gi:119714272; M.l., gi:13471039; MtCASTOR, gi:57494651. Bar: number of amino acid substitution per site between two sequence.
1.0
Angiosperm Eubacteria
Bryophyte
Angiosperm
Angiosperm
M.t.DMI1 P.s.SYM8
M.t. CASTOR
100
L.j.CASTOR
L.j.POLLUX
V.v.2
O.s.1
A.t.1
V.v.1
O.s.2 A.t.2
A.t.3 S.a.
N.sp. M.l.
P.p.
O.s.3
99
100
60
99
100
99
100 64 100
100
100
100
99
33
Phylogenetic analyses using a character-based method, the parsimony analysis,
were conducted with PHYLIP to ascertain the relationship of CASTOR and POLLUX to
other homologous proteins. Analyses of full-length proteins from plants, moss and
bacterial species yielded four well-supported clades corresponding to three orthologous
groups of plant proteins and a single group of bacterial proteins (Figure 4).
CASTOR and POLLUX have 28% and 31% identity, respectively, with the
deduced partial protein Physcomitrella patens, which is consistent with an origin in the
nonvascular plants. Taken together with the absence of homologs in the fungal, animal
and green algal lineages, these results suggest that the CASTOR and POLLUX proteins
represent a plant specific innovation that potentially arose near the base of the land plant
lineage. The relationship between the prokaryotic sequence and the eucaryote derivative
is difficult to deduce from the limited prokaryotic sequence data. On the one hand, the
weak sequence identity between the bacterial and moss proteins (< 20%) suggests that the
plant and bacterial proteins date back to a primordial precursor. On the other hand, the
absence of homologs in archaea and green algae may suggest a horizontal gene transfer
between an ancient plant genome(s) and a bacterial species.
CASTOR and POLLUX encode predicted integral membrane proteins of 853 and
917 amino acids, respectively, with at least four transmembrane regions (Figure 5A).
Overall, the two proteins are very similar to each other with the exception of the N-
terminal regions which are more divergent (Figure 5A). Other than these general features,
CASTOR and POLLUX lack similarity to functionally characterized proteins. The
middle region of each protein includes a domain of unknown function (DUF) 1012 found
in both eukaryotes and bacteria (Figure 5A). The POLLUX DUF1012 domain overlaps
with a region of low, but global similarity to the TrkA-N domain, a nicotinamide adenine
dinucleotide (NAD) binding domain, found in a wide variety of proteins, including
potassium channel, phosphoesterases and various other transporters (Schlosser et al.,
1993). Similarly, the CASTOR DUF1012 domain overlaps with a predicted NAD(P)
binding domain. Overlapping with the two transmembrane domains (TM) and the
putative NAD binding domain, a PRK10537 domain is predicted in both CASTOR and
POLLUX (Figure 5A).
34
Figure 5. Sequence analyses of CASTOR and POLLUX. (A) Prediction of conserved domains in CASTOR (853 a.a.) and POLLUX (918 a.a.) were conducted with the programs: Membrane Protein Explorer 3.0 to predict transmembrane domains (TM), NCBI/BlastP to predict domain of unknown function (DUF) 1012, TrkA-N (PF02254) and PRK10537, EMBL/InterProScan to predict NAD(P)bd, and FUGUE to predict the partial sequence-structure homolog Methanobacterium thermoautotrophicum calcium-gated potassium channel MthK (027564). (B) Alignment of the filter and inner helical region of the MthK channel with the structural homologous region of CASTOR, POLLUX and plant homologues. This region of MthK forms the core of the pore predicted in (C). (C) X-ray crystal structure of the ion pore region of the MthK channel (Protein Data Bank code1LNQ), and a model of the homologous region in CASTOR. The filter region is coloured orange: inner helices are coloured yellow. Residues Gly61 and Gly63 in the MthK structure, and the corresponding Ser and Asn residues in CASTOR, are shown in space-filling mode. Conserved residues Gly83 and Ala88 in the inner helix are highlighted in green. (D) Model of the ion pore region of one CASTOR monomer. Amino acid residues constituting the selectivity filter are labeled with the carbonyl oxygen backbone facing the pore. Red: oxygen atoms, blue: amino groups, white: carbon atoms.
35
The PRK10537 domain is typically conserved in proteobacteria voltage-gated potassium
channels. However, CASTOR and POLLUX sequences do not include any voltage sensor
domains which are characterized by positively charged residues periodically aligned in
transmembrane segments (Bezanilla, 2000). Search based on sequence-structure
homology using FUGUE (Shi et al., 2001) resulted in low structural matches to the
calcium-gated potassium channel of Methanobacterium thermoautotroficum, MthK, for
which a crystal structure has been resolved (Jiang et al., 2002a) (Figure 5A). CASTOR
and POLLUX share a structural homology only with a short stretch of MthK sequence
covering the pore region, which includes the filter and the inner helix (Figure 5B).
Alignment of the pore region of MthK with CASTOR, POLLUX and plant homologues
revealed that two important residues of the inner helix, Gly83 and Ala88, are conserved
(Figure 5B). Both Gly83 and Ala88 are involved in the mechanics of MthK pore gating.
The Gly83 confers the flexibility to the helix to bend and Ala88 constitutes the narrowest
point in the intracellular entryway (Jiang et al., 2002b). Despite these similarities between
MthK and CASTOR-POLLUX, the potassium signature, TxGYG (Heginbotham et al.,
1994), which confers to MthK its potassium selectivity, is not conserved (Figure 5B).
Homology modelling of the filter region of CASTOR indicates that the differences could
result in a more helical backbone, and this would modify the diameter and the
electrostatic properties of the pore (Figure 5C). However, the carbonyl oxygen backbone
facing the pore is conserved between MthK and CASTOR, suggesting a cation selectivity
(Figure 5D). Nevertheless, the absence of the highly conserved potassium signature
sequence and the low structural homology between either protein and MthK left the
function totally unclear and prompted us to study the biochemical and
electrophysiological properties of CASTOR and POLLUX.
2.3 Nuclear localization of CASTOR and POLLUX
CASTOR and POLLUX are predicted to carry potential chloroplast transit peptides of 69
and 57 amino acids at their N-termini (TargetP scores 0,953 and 0,959), respectively.
Although TargetP (http://www.cbs.dtu.dk/services/TargetP/) predicted a plastid
36
Figure 6. Localization in tobacco leaf epidermal cells of CASTOR and POLLUX. (A) Representation of CASTOR, POLLUX and truncated versions. TM, Transmembrane domain; yellow line, nuclear localisation signals of CASTOR (HRRR) and POLLUX (left to right PPLKKTK, RKRRP, PLKKRVA); black boxes, potential chloroplast transit peptide. (B) and (D) Microscopic pictures of leaf epidermal cells expressing a POLLUX:GFP fusion and (D) and (E) a CASTOR:GFP fusion under the control of a double cauliflower mosaic virus 35S promoter (2xP35S). (F) to (I) Confocal laser scanning images of tobacco leaf epidermal cells transiently expressing a POLLUX:GFP fusion and (J) to (M) a CASTOR:GFP fusion under the control of a single cauliflower mosaic virus 35S promoter (1xP35S). (F) and (J) GFP fluorescence, (G) and (K) 4´, 6-diamidino-2-phenylindole (DAPI), (H) and (L) GFP, DAPI and Bright field merged, (I) and (M) Confocal microscopic images of individual optical sections of GFP fluorescence. (P) to (S) Microscopic pictures of leaf epidermal cells expressing the TM1POL:RFP fusion, and (T) to (W) a TM1CAS:RFP fusion under the control of a single cauliflower mosaic virus 35S promoter (1xP35S). (P) and (T) RFP fluorescence, (Q) and (U) control leaf epidermal cells expressing N-terminal of the signal peptide of spinach ferredoxin NADP(H) oxidoreductase, a thylakoid bound enzyme (FNR) fused to GFP, (R) and (V) bright field, (S) and (W) RFP, GFP and Bright field merged. Bars = 10 mm. (N) Detection of 2xP35S:CASTOR:GFP and 2xP35SPOLLUX:GFP, and (O) 1xP35S:CASTOR:GFP and 1xP35S: POLLUX: GFP fusion proteins in tobacco by immunoblot. (N) Total protein extracts from leaves expressing GFP alone (1), POLLUX:GFP (2) and CASTOR:GFP (3). (O) Total protein extracts from leaves expressing POLLUX:GFP (1) and CASTOR:GFP (2-3) and empty vector (4). Top panel: immunoblot with anti-GFP. Lower panel: Coomassie stained blot.
37
localization for CASTOR and POLLUX, Wolf PSORT and Prosite
(http://wolfpsort.seq.cbrc.jp/; http://www.expasy.org/prosite/) predicted three and one
putative nuclear localization signal (NLS) within CASTOR and POLLUX sequences,
respectively (Figure 6A). Moreover, in adition to the predicted NLS, the subprediction
methods SVMaac of MultiLoc/TargetLoc (http://www-bs.informatik.uni-tuebingen.de/
Services/MultiLoc/) reveals that CASTOR and POLLUX may have the properties to be
targeted in plastid (SVMaac scores, CASTOR; 0.94 and POLLUX; 0.88) or in the ER
(SVMaac scores, CASTOR; 0.68 and POLLUX; 0.63).
In order to determine the localization of CASTOR and POLLUX in planta, both
CASTOR and POLLUX fused to the green fluorescent protein (GFP) were transiently
expressed under the control of cauliflower mosaic virus 35S promoter (P35S) via particle
bombardment in onion epidermal and pea root cells (Imaizumi-Anraku et al., 2005). In
these expression systems, both CASTOR and POLLUX displayed plastid localization
patterns (Imaizumi-Anraku et al., 2005). The plastid localization was further confirmed
with CASTOR and POLLUX expressed under the control of double P35S in Nicotiana
benthamiana leaves via Agrobacterium tumefaciens mediated transformation (Figure 6R
to 6U). As a marker for the plastid localization, the transit peptide of spinach ferredoxin
NADP(H) oxidoreductase, a thylakoid bound enzyme (FNR) (Lohse et al., 2005) was
cloned and fused to the GFP (Figure 6C and 6G). Surprisingly, CASTOR and POLLUX
expressed under the control of a single P35S in Nicotiana benthamiana leaves via
Agrobacterium tumefaciens mediated transformation displayed a localization in the
nuclear envelope (Figure 6J to 6Q). In all experiments, the expression of the full length
CASTOR and POLLUX was detected by immunoblot with anti-GFP excluding a possible
localization of truncated proteins or free GFP (Figure 6V and 6W). To assess whether
CASTOR and POLLUX N-termini sequences containing both the predicted chloroplast
transient peptides and first NLS would localize the proteins in the plastid or in the nuclear
periphery, we generated additional fusion between the red fluorescent protein (RFP) and
N-terminal fragment encompassing the first 139 amino acids and 208 amino acids of
CASTOR and POLLUX (TM1CASTOR:RFP and TM1POLLUX:RFP), respectively (Figure
6A). In Nicotiana benthamiana leaves, for both TM1CASTOR:RFP and TM1POLLUX:RFP
expressed under a single P35S, the red fluorescence was observed at the periphery of
nuclei (Figure 6B and 6F). No colocalization of either TM1CASTOR:RFP or
38
TM1POLLUX:RFP with the plastid control (FNR:GFP) could be observed (Figure 6E and
6I), confirming an exclusive localization of TM1CASTOR:RFP and TM1POLLUX:RFP in the
nuclear rim. These results indicate that the N-termini sequences of CASTOR and
POLLUX contain active nuclear localization signals sufficient to target the protein to the
nuclear rim under the expression straight of a single P35S. However, when
CASTOR:GFP and POLLUX:GFP are expressed under the control of double P35S, the
green fluorescence was observed in plastids which all together demonstrate that entirely
different localization patterns are detectable in transient assays.
In order to assess the localization in L. japonicus root cells and to exclude
possible artefacts associated with the transgenic expression of fusion proteins, we
targeted the endogenous proteins using specific antibodies. Rabbit polyclonal antibodies
were raised against hydrophilic peptides of CASTOR and POLLUX. In immunoblots, the
purified CASTOR antibodies allowed a specific detection of CASTOR (Figure 7A),
whereas the POLLUX antibody was unspecific (data not shown). The immunogold
labelling with anti-CASTOR was performed on wild type L. japonicus and on castor-12,
a mutant carrying a single point mutation leading to a premature stop codon at position
W93 (Table 2). By electron microscopical analysis of immunogold labelled sections, a
strong and specific accumulation of gold particles was observed at the position of the
nuclear envelope of the wild type. This labelling was absent in samples of the castor-12
mutant (Figure 7B to 7E). The separation of the membranes was not well preserved by
the immunolocalization compatible embedding method used. Therefore, we could not
determine whether CASTOR resides in the inner or outer membrane of the nuclear
envelope, or in both. Only non-specific background labelling was observed in plastids in
both wild type and castor-12 (Figure 7B). Collectively, these data provide evidence for a
localization of CASTOR in the nuclear envelope.
39
Figure 7. Immunogold localization of CASTOR in Lotus japonicus with anti-CASTOR. (A) Immunoblot detection with anti-CASTOR in total extract of transformed roots of Lotus japonicus mutant castor-12 (W93*). 1; castor-12 complemented with P35S:CASTOR via A. rhizogenes hairy root transformation, 2; castor-12 transformed roots with empty vector as negative control, lower panel; loading shown by Coomassie staining of the blot. (B) Relative density of gold particles in different cellular compartments of L. japonicus Gifu wild type and mutant castor-12 roots after immunogold labelling with anti-CASTOR. (C) to (E) Electron microscopic pictures of L. japonicus Gifu wild type nucleus (C) and castor-12 nucleus (E). (D) Zoom of the black box of picture (C). Dashed white line: delimitation of the nuclear envelopes. c cytoplasm; cw, cell wall; n, nucleus; er, endoplasmic reticulum. Black arrowheads mark positive immunodetection of CASTOR. White arrowheads mark background labelling in the nuclear matrix.
c n
cw
er
1 µm
C D
200 nm
n
c er
A
83
KDa 175
2 1
Anti-CAS Cellular
compartment Relative labelling density
Cell wall Plasma membrane Vacuole Plastids Mitochondria Nuclear envelope Nuclear matrix ER
< 1 < 1 < 1 5 +/- 3 16 +/- 10 156 742 +/- 1000 158 +/- 100 < 1
Wild type castor-12
B
< 1 < 1 < 2 8 +/- 2 50 +/- 30 < 1 350 +/- 100 < 1
E
200 nm
n
c
40
2.4 Homodimerization of CASTOR and POLLUX
One common feature of ion channels is their organization in homo- or hetero-tetrameric
complexes of identical or similar subunits, respectively (Christie, 1995). We checked the potential of the C-terminal soluble domains of CASTOR (cCASTOR; H322-E853) and POLLUX (cPOLLUX; H386-D917) to form homo- or heteromers. In yeast-two-hybrid
assays, cCASTOR and cPOLLUX were each capable of self-interaction (Figure 8A). The interaction between POLLUX and CASTOR was only observed using cCASTOR fused to the GAL4 activator domain (AD) and cPOLLUX to the GAL4 binding domain (BD), but
never when cCASTOR and cPOLLUX were fused to BD and AD, respectively (Figure 8A). To assess the specificity of AD:cCASTOR interaction, we mutated cCASTOR according to a series of castor mutant alleles carrying a point mutation or deletion (Table 2). Five ccastor
mutant versions, ccastor-1 (∆LA 479-480), ccastor-7 (V598I), ccastor-13 (D444N), ccastor-17 (R590H) and ccastor-28 (D537G), were tested for cCASTOR dimerization in yeast-two-hybrid assays. AD:cCASTOR and BD:ccastor-1, expressed in yeast (Figure 8B), did not
interact indicating that interactions of AD:cCASTOR with either BD:cCASTOR or BD:cPOLLUX were specific (Figure 8C). Furthermore, the AD:cPOLLUX dimerization was tested with two mutant versions, cpollux-7 (V531E) and cpollux-8 (W781*) (Table 3),
(Figure 8D). AD:cPOLLUX and BD:cpollux-8, expressed in yeast (data not shown), did not interact, demonstrating the specificity of POLLUX dimerization, as well as the requirement of a full C-terminal sequence for interaction. All together, these results
demonstrate that cCASTOR and cPOLLUX can homo-dimerized. Furthermore, the homo-dimerization requires their C-termini domains.
To address the question of CASTOR and POLLUX hetero- or homo-dimerization
in planta, we used a bimolecular fluorescence complementation (BiFC) approach in tobacco epidermal cells (Figure 9A). The full length CASTOR and POLLUX were fused to either the N- or C-terminal half of the yellow fluorescent protein (YFP) gene. The expression of all
fusion proteins was confirmed by immunoblots (Figure 9B). All BiFC CASTOR and POLLUX fusion constructs complemented L. japonicus castor or pollux mutant alleles, respectively, suggesting that these constructs are functional (Table 5). In line with the yeast-
two-hybrid and localization results, the YFP fluorescence due to the full-length CASTOR or POLLUX self-interactions was clearly observed around the nucleus and the latter was abolished by the castor-1 mutation (Figure 9A). Although AD:cCASTOR and
41
BD:cPOLLUX interact in yeast, the heterodimerization of CASTOR and POLLUX, both tested either in fusion with the N- or C-terminal half of the YFP, was never seen in tobacco epidermal cells (Figure 9A, data not shown). All together, these observations suggest that, in
planta, CASTOR and POLLUX form two distinct complexes for which the assembly involves the C-terminal region of the proteins.
Figure 8. Yeast-two-hybrid assay for interactions of cCASTOR and cPOLLUX. (A) Interaction assays were performed in the yeast strain AH109 cotransformed with the bait (BD) and prey (AD) vectors containing either cCASTOR (H322-E853) or cPOLLUX (H386-D917). The cotransformants and interacting partners were analysed on synthetic dropout nutrient medium lacking leucine and tryptophan (-LW), and lacking leucine, tryptophan, adenine and histidine (-LWAH), respectively. (B) Expression analyses of BD:cCASTOR∆LA 479-480 by immunoblot. Total extract from yeast transformed with empty vector (1) and vector expressing BD: cCASTOR∆LA 479-480 (2). (C) Yeast-two-hybrid interaction analyses of ccastor mutant versions. All tested mutations originate from symbiosis-defective castor mutant alleles; castor-1 (∆LA 479-480), castor-7 (V598I), castor-13 (D444N), castor-17 (R590H) and castor-28 (D537G) (Table 2). (D) Yeast-two-hybrid interaction analyses of cpollux mutant versions. All tested mutations originate from symbiosis-defective pollux mutant alleles; pollux-7 (V531E) and pollux-8 (W781*) (Table 3). Dark line; single amino acid change, White line; deletion.
B
83
KDa
62
1 2
A
BD AD
cCASTOR
cPOLLUX
-LWAH -LWAH -LW -LW
cCASTOR cPOLLUX anti-BD
C AD:cCASTOR
-LWAH -LW
BD:ccastor-7 (V598I)
BD:ccastor-17 (R590H)
BD:ccastor-28 (D537G)
BD:ccastor-1 (∆LA 479-480)
BD:ccastor-13 (D444N)
D AD:cPOLLUX
-LWAH -LW
BD:cpollux-7 (V531E)
BD:cpollux-8 (W781*)
42
Figure 9. Formation of CASTOR and POLLUX homocomplexes in planta. (A) BiFC analysis of CASTOR (CAS) and POLLUX (POL) interaction in transiently transformed N. benthamiana leaf epidermal cells. CASTOR and POLLUX driven by the cauliflower mosaic virus 35S promoter were fused to the N-terminal half of yellow fluorescent protein (YFP) and a c-myc tag (N), or to the C-terminal half of YFP and a HA tag (C). Self-interactions between CASTOR∆LA-479-480 (cas-1) and the nuclear localized transcription factor bZIP63 (BZIP) from A. thaliana were used as negative and positive controls, respectively. The experiment was visualized with an inverted epifluorescence microscope. Left panel: YFP fluorescence, right panel: bright field and YFP fluorescence merged. Bars = 20 µm. (B) Protein expression analysis by immunoblot. The protein extracts from leaves transiently transformed with empty vector (1), or vectors expressing POL:C and POL:N (2), CAS:C and CAS:N (3), cas-1:N and cas-1:C (4), CAS:N and POL:C (5), CAS:N and POL:C (6) were analyzed. Expected sizes of POLLUX: 102 kDa, CASTOR: 95 kDa, N-terminal half of the yellow fluorescent protein fused to c-myc tag (N): 18 KDa, C-terminal half of the yellow fluorescent protein fused to HA tag (C): 10 kDa. Top panel: immunoblot. Lower panel: Coomassie stained blot documenting protein loading.
Table 5. Restoration of Root Nodule Symbioses in A. rhizogenes-transformed root systems of L. japonicus mutants pollux-2 and castor-12 with the different POLLUX and CASTOR split YFP versions, respectively.
Plant genotype Transgene Fraction of nodulated plants
Nodule / nodulated plants
wild type 35S:N 32/49 4.6 wild type 35S:C 28/30 4.1 pollux-2 35S:N 0/89 0 pollux-2 35S:POL:N 30/57 4.2 pollux-2 35S:POL:C 43/72 3.8 castor-12 35S:N 0/64 0 castor-12 35S:CAS:N 59/85 3.4 castor-12 35S:CAS:C 54/78 5.1
120 112
A CAS:N / CAS:C
POL:N / POL:C
CAS:N / POL:C cas-1:N / cas-1:C
BZIP:N / BZIP:C B 120 112 105
c-myc+HA c-myc HA 1 2 3 4 5 6 KDa
43
2.5 Expression of POLLUX and DMI1 under the control of P35S restores the
nodulation phenotype of castor-12 mutant
Both CASTOR and POLLUX are two complexes formed by homo-units assembling. The
expression level of CASTOR and POLLUX mRNA in root, nodule, leaf, seed pod and flower bud was examined by quantitative RT-PCR (Imaizumi-Anraku et al., 2005). CASTOR and POLLUX were both constitutively expressed. M. loti inoculation or Nod-
factor treatment did slightly repress the expression of CASTOR and POLLUX genes
during the first two days (Imaizumi-Anraku et al., 2005). To determine the spatial expression pattern of CASTOR and POLLUX in L. japonicus roots before and after M. loti
inoculation, we used the β-glucuronidase (GUS) reporter gene fused with a 5´- upstream region of 2.3kb and 2.8kb for CASTOR and POLLUX, respectively. In A. rhizogenes transformed roots expressing the GUS reporter gene driven by either CASTOR or POLLUX
promoters, the patterns of GUS activity were similar in all roots with the strongest staining at the growing tips (Figure 10). No differences in the expression pattern of either construct were observed before and after 24h inoculation with M. loti.
The similar sequences, expression patterns and localization of CASTOR and POLLUX suggest that they have similar roles. However, mutations in either castor or pollux alone lead to symbiosis-deficiency. Taken together, these data raise the hypothesis that a
certain threshold of CASTOR and POLLUX is required for symbiosis. In order to test this, we expressed CASTOR and POLLUX under the control of a single P35S in castor-12 and pollux-5 mutant alleles via A. rhizogenes transformation system. Pollux-5 contains a
nonsense mutation W321* leading to a truncated protein with three transmembrane domains while castor-12 (W93*) does not retain any (Table 2 and 3). The P35S:CASTOR construct complemented castor-12 but not pollux-5 (Figure 11A and 11B). However, the
P35S:POLLUX construct complemented pollux-5 as well as castor-12 (Figure 11A and 11B). This result clearly demonstrates that the homocomplex POLLUX can compensate the loss of CASTOR when expressed under the control of P35S, which strongly suggests that
POLLUX has a biochemical function similar to CASTOR. In line with the BiFC results, these data imply also that POLLUX localizes to the same nuclear membrane as CASTOR in planta.
44
Figure 10. Expression patterns of CASTOR and POLLUX promoter:GUS fusions in L. japonicus roots before and after inoculation with Mesorhizobium loti. A. Rhizogenes-transformed root systems of L. japonicus expressing the GUS reporter gene fused to the CASTOR and POLLUX promoters (PCAS:GFP:GUS and PPOL:GFP:GUS) were examined for GUS activity without and with M. loti strain R7A 24h after inoculation. Bars = 200 mm.
- M. loti
- M. loti
+ M. loti
+ M. loti
PCAS:GFP:GUS
PPOL:GFP:GUS
45
Figure 11. Restoration of root nodule symbiosis in A. Rhizogenes-transformed root systems of L. japonicus mutants. (A) Pictures of transgenic hairy roots three weeks after inoculation with Mesorhizobium loti R7A. The T-DNA carried the green fluorescent protein (GFP) as a marker gene. Left panel; GFP fluorescence, right panel; bright field. Bars = 1 mm. (B) Nodulation of transgenic roots. The fraction of nodulated plants and the number of nodules per nodulated plants were determined three weeks after inoculation with the Mesorhizobium loti R7A strain. The standard deviations are indicated between brackets.
P35S:POLLUX
P35S:CASTOR
Plant genotype Transgene Fraction of nodulated plants
Nodules/ Nodulated plant
Wild type
castor-12
castor-12
castor-12
pollux-5
pollux-5
pollux-5
marker only
marker only
marker only
P35S:CASTOR
P35S:CASTOR
P35S:POLLUX
P35S:POLLUX
23/23
0/39
19/20
14/18
0/12
31/33
0/23
3.43 (+/- 1.0)
0
3.1 (+/- 0.8)
2.5 (+/- 0.5)
0
3.75 (+/- 1.1)
0
pollux-5
pollux-5
castor-12
castor-12
A
B
46
In order to determine whether the M. truncatula DMI1 (Does not Make Infection 1) (Ane et al., 2004), the POLLUX putative ortholog, could restore the nodulation phenotype of the castor and pollux mutant, transgenic roots of these plants carrying the fusion construct
DMI1:GFP under the control of a single P35S were generated and inoculated with the M.
loti R7A strain. Similarly to POLLUX, DMI1 fully restored the nodulation phenotype of castor-12 and pollux-1 mutant (Figure 12A and 12B). Furthermore, in line with the
previous report showing DMI1 localized around the nucleus (Riely et al., 2007), DMI1 localizes at the nuclear periphery in castor-12 and pollux-1 mutants (Figure 12C).
Plant genotype Transgene Fraction of nodulated plants
Nodules/ Nodulated plants
Wild type marker only 20/20 5 (+/- 2) castor-12 marker only 0/30 0 castor-12 P35S:DMI1:GFP 15/15 4.2 (+/- 2) pollux-1 marker only 0/22 0 pollux-1 P35S:DMI1:GFP 40/40 4.1 (+/- 3)
Figure 12. Restoration of root nodule symbiosis in A. rhizogenes-transformed root systems of L. japonicus mutant with the M. truncatula DMI1 gene under the control of single P35S. (A) Pictures of transgenic hairy roots four weeks after inoculation with the Mesorhizobium loti R7A strain. The T-DNA carried the Discosoma sp. red fluorescent protein (DsRed) as a marker gene. Left panel; DsRed fluorescence, right panel; bright field. Bars = 2 mm. (B) Nodulation of transgenic roots. The fraction of nodulated plants and the number of nodules per nodulated plants were determined four weeks after inoculation with the Mesorhizobium loti R7A strain. The standard deviations are indicated between brackets. (C) Confocal laser scanning images of A. rhizogenes-transformed root systems of L. japonicus pollux-1 and castor-12 mutant with either DMI1:GFP fusion under the regulation of P35S (Riely et al., 2007) (bars = 30 µm) or GFP under the regulation of P35S (bars = 35 µm).
B
A C
castor-12
pollux-1
castor-12
pollux-1
castor-12
pollux-1
P35S:DMI1:GFP P35S:GFP
47
Collectively, these data confirm that POLLUX and its M. truncatula ortholog
DMI1 have a function similar to CASTOR. Furthermore, the complementation of castor
mutant with POLLUX highlights an identical localization of CASTOR and POLLUX in
L. japonicus.
2.6 Functional characterization
The amino acid sequence of CASTOR and POLLUX filters did not match to any known
conserved signature, giving no information concerning ion selectivity. However, the
model of CASTOR predicted a decoration of the pore with carbonyl oxygen atoms
suggesting a cation selectivity (Figure 5D). We therefore tested, via yeast
complementation and electrophysiological approaches, the hypothesis that CASTOR and
POLLUX act as cation channels.
2.6.1 Yeast complementation assays
2.6.1.1. cch1∆mid1∆ and trk1∆ yeast mutants complementation assays
CCH1 and MID1 are components of a high-affinity Ca2+ permeable channel (Fischer et
al., 1997; Iida et al., 1994), whereas TRK1 is a high-affinity K+ transporter conferring to
the yeast K+ uptake system a higher K+/Na+ discrimination under Na+ stress (Gaber et al.,
1988). The CCH1/MID1 complex is involved in the generation of calcium influx in
response to various biotic or abiotic factors; budding, alpha-mating pheromone, cold or
iron stress (Fischer et al., 1997; Peiter et al., 2005). The cch1Δmid1Δ mutant also displays
reduced tolerance to monovalent cations such as Li+ (Paidhungat and Garrett, 1997),
whereas the absence of TRK1 confers to trk1Δ mutant a higher sensitivity to Na+ (Calero
et al., 2000; Gaber et al., 1988).
In order to check if CASTOR and POLLUX could complement the reduced
tolerance to lithium for cch1Δmid1Δ mutant or to sodium for trk1Δ mutant, both proteins
tagged to a herpes simplex virus protein, VP16, were expressed under the regulation of
the S. cerevisiae alcohol dehydrogenase 1 promoter. POLLUX could be detected in the
48
microsomal fraction by immunoblot with anti-VP16 (Figure 13A and 14B), whereas the
full-length CASTOR expressed with different vectors could either not be detected or
appears to be cleaved in the yeast mutants (Figure 13A and 14B).
The expression of POLLUX did not suppress the phenotypes of cch1Δmid1Δ or
trk1Δ strains (Figure 13B and 13C). In contrast, expression of POLLUX in trk1Δ mutant
reduced the mutant growth by 20 % (Figure 13C). These data demonstrate that POLLUX
increases the sodium sensitivity of the mutant.
Figure 13. Complementation of the yeast double mutant cch1∆mid1∆ and trk1∆ strains. (A) Expression analyses of CASTOR and POLLUX in the yeast cch1∆mid1∆ mutant JK9-3 da strain. Upper panel: immunoblot detection with anti-VP16, lower panel: Coomassie blue staining of the membrane. Protein extracts from cch1∆mid1∆ mutant transformed with the empty vector pTMBV4 (1), pTMBV4 vector carrying CASTOR (2) or POLLUX (3) under the regulation of the S. cerevisiae alcohol dehydrogenase 1 promoter and fused to the tag VP16 (Hepres simplex VP16 transactivator). m; microsomal fraction, s; soluble fraction. (B) Growth assay of cch1∆mid1∆ mutant JK9-3 da strain transformed with either CASTOR or POLLUX on yeast synthetic defined medium lacking leucine (SD-L) containing 200 mM LiCl. Growth of JK9-3 da wild type strain transformed with the empty vector pTMBV4 (1), growth of cch1∆mid1∆ mutant JK9-3 da strain transformed with pTMBV4:CASTOR (2), pTMBV4:POLLUX (3) or empty vector pTMBV4 (4). (C) Growth of NaCl sensitive mutant trk1∆ on SD-L medium supplemented with different concentrations of NaCl. ; trk1∆ mutant transformed with pTMBV4:POLLUX, ; trk1∆ mutant transformed with empty vector pTMBV4, ; wild type transformed with empty vector pTMBV4. Standard errors of the means from 3 experiments.
2.6.1.3 MAB 2d yeast mutant complementation assays
In order to check whether POLLUX could trigger a potassium influx, we assayed POLLUX for complementation of a yeast mutant, MAB 2d, in which the main potassium importers
B
1 2 3 4
- +
LiCl 200 mM
C A
s s s m m m 1 2 3
175 83 62 83 62
KDa
49
and exporters are deleted (trk1Δ trk2Δ ena1-4Δ nha1Δ) (Kolacna et al., 2005; Maresova and Sychrova, 2005; Rodriguez-Navarro, 2000). The deletion of both the potassium efflux and influx systems enables the MAB 2d strain to grow on acidic minimal medium supplied
with otherwise toxic potassium concentrations of 600 mM (Kolacna et al., 2005). A second aspect of the potassium-uptake deficient phenotype is the inability of the mutant to grow at potassium concentrations from 10 to 50 mM (Kolacna et al., 2005). The expression of
POLLUX under the regulation of the Saccharomyces cerevisiae alcohol dehydrogenase promoter in the strain MAB 2d impaired the growth of the mutant at potassium concentrations between 300 and 600 mM KCl, and promoted the growth on medium
containing between 10 to 50 mM KCl (Figure 14). These results demonstrate that in MAB 2d, POLLUX acts as a channel triggering an influx of potassium which can be detrimental or advantageous for the yeast depending on the concentration of potassium available in the
medium.
Figure 14. Complementation of MAB 2d mutant. (A) Growth of MAB 2d cells transformed with pDL2:POLLUX (POL) under the S. cerevisiae alcohol dehydrogenase 1 promoter, or the empty vector (pDL2) at indicated KCl concentrations on minimal medium pH 4. (B) CASTOR and POLLUX were expressed in the yeast strain MAB2d. Only degradation products of CASTOR were detected, while a single band corresponding to the predicted size of the POLLUX full length protein (102 kDa) was observed
All together, these data indicate that POLLUX could trigger both an influx of potassium
and an influx of sodium inside the yeast, which suggests that POLLUX is a non selective
cation channel localized to the yeast plasma membrane. The attempts to confirm the
localization of POLLUX in yeast were unsuccessful. However, in planta, POLLUX
under the control of a different promoter could end up in a different sub-cellular
A
10 30 50 100 200 300 400 600 KCl (mM)
pDL2:POL
pDL2
83
62
kDa pDL2
pDL2 CAS:HA
pDL2 POL:HA
B
50
membrane. Therefore, considering our results, the plasma membrane localization of a
fraction of POLLUX in yeast cannot be excluded.
2.6.2 Electrophysiological analysis
2.6.2.1 CASTOR is a cation channel
In order to determine whether CASTOR is an ion channel, we expressed CASTOR using an
in vitro transcription-translation system (Figure 15A and 15B). The protein was purified by selective solubilization (Figure 15C and 15D), denatured and subsequently reconstituted into liposomes by dialysis. Proteoliposomes were then fused into planar lipid bilayers
(Morera et al., 2007). The modeling of CASTOR filter based on the structural homology with MthK predicted a decoration of the pore with carbonyl oxygen atoms from the peptide backbone, suggesting selectivity for cations (Figure 5D). To test this prediction
experimentally, we checked the permeability of CASTOR for physiologically relevant cations over anions. In assymetrical KCl or NaCl solutions of 250 and 20 mM, we recorded the reverse potential of CASTOR which corresponds to the membrane potential at which the
net flow of ions from one reservoir to the other is zero (Figure 16A and Table 6A). The calculation of the permeability ratio was based on the Goldman-Hodgkin-Katz current equation (Goldman, 1943; Hodgkin and Katz, 1949). The permeability ratios PK+/PCl- of
6.09, PNa+/PCl- of 1.66 and PCa2+/PCl- of 0.52 indicate a preference of CASTOR for potassium (Table 6A). To test if the preference for potassium was not influenced by a conformational change of the filter due to the presence of only one cation in excess
(Valiyaveetil et al., 2006), we performed competition assays between K+, Ca2+ and Na+ (Table 6B). Solutions of K+ and Ca2+ or K+ and Na+ were applied to the left and right side of the planar lipid bilayer, respectively. The permeability ratios PK+/PNa+ and PK+/PCa2+,
indicated a 2-fold preference for K+ in comparison to Na+ and Ca2+. These results demonstrate a moderate preference of CASTOR for K+ over Na+ and Ca2+, which suggests that CASTOR is a poorly selective cation channel.
51
Figure 15. Cell free expression of CASTOR, castor-2 and POLLUX. (A) Autoradiography of CASTOR (1) and POLLUX (2), exp-ressed in a cell free system and labelled radioactively with L-[35S]methionine. (B) and (C) Coomassie Blue staining of pro-tein samples analyzed by SDS/PAGE in 6% (v/v) tricine gel. (B) Total extract from the cell free expression system. S, soluble fraction; P, pelleted fraction; black arrow, CASTOR; asterisk, castor-2. (C) Purification of CASTOR by selective solubilization. 5 µL of soluble fraction were analysed after centrifugation of the total cell free CASTOR expressed samples (lane 1), washing in sodium phosphate buffer pH7.2 (lane 2 and 3), washing in sodium phosphate buffer pH7.2 and 2% (v/v) MEGA-9 1h (lane 4 and 5), washing in sodium phosphate buffer pH7.2 and 0,1% (v/v) SDS 1h (lane 6). (D) Immunoblot analyses with anti-CASTOR using 2 µg of solubilized CASTOR. 1; Total protein of POLLUX expressed in cell free system, 2; CASTOR purified by selective solubilization.
Castor-2 carries a single nucleotide replacement which leads to the substitution of alanine by threonine at the position 264 (Figure 17 and Table 2). The castor-2 mutant is defective in Ca2+ spiking (Naoya Takeda unpublished data) and both root nodule and AM
symbioses (Table 2). The mutated alanine is positioned at the hinge of the selectivity filter, which could alter the selectivity of the channel (Figure 17). To test this hypothesis, we determined the reverse potential (Figure 18A) and subsequently the permeability ratio of
castor-2 for K+ and Na+ (Table 6A). We observed that castor-2 was equally permeable to Na+ and K+ (Table 6). This result indicates that the A264T mutation alters the selectivity of the pore.
B A 1 2
175
83
62 48
KDa S P S P CASTOR
S P castor-2 POLLUX
175
83
KDa
*
C 175
83
62
1 2 3 4 5 6 KDa D 175
83
62
1 2 KDa
coomassie
coomassie
immunoblot
autoradiogram
52
Table 6. Reverse potential (Erev) and permeability ratios (PX/PCl-) for CASTOR and castor-2 (A), and reverse potential and permeability ratios PK+/PX for CASTOR (B). A
CASTOR castor-2
Ion (X) Erev (mV) PX/PCl- Erev (mV) PX/PCl- K+ 36 +/- 2 (n=40) 6.0 a 28 +/-2 (n=48) 3.7 a Na+ 11 +/- 1 (n=8) 1.6 a 25 +/-3 (n=13) 3.2 a Ca2+ -29+/- 2 (n=13) 0.52 a -31 +/-2 (n=19) 0.6 a
B Ion (X) Erev (mV) PK+/PX
Na+ 17.9 +/- 2 (n=5) 2.0 a Ca2+ 0.4 +/- 1 (n=17) 2.0 b
a Permeability ratios calculated from reverse potentials using equation: Erev= RT/zF x ln(PK+[K]o/PNa+[Na]i) b Permeability ratios calculated from reverse potentials using equation: Erev= RT/F x ln(√((4PCa2+[Ca]o/PK+[K]i)+¼) – ½) whith (RT/F= 25.26 mV, Z= 1). The junction potential correction; Agar bridge 2M KCl, (A) NaCl (250 mM/20 mM,) Vm = Vcmd + 2.3 mV; KCl (250 mM/20 mM) Vm = Vcmd + 1.2 mV; CaCl2 (125mM/10mM) Vm = Vcmd + 3.1 mV (B) KCl-NaCl: Vm=V-1.1 mV; KCl-CaCl2: Vm=V-2.3 mV.
Figure 16. Electrophysiological characterization of CASTOR. (A) Selectivity of the CASTOR channel. Voltage ramps (10 mV/s) were applied to bilayers containing one copy of CASTOR at asymmetrical electrolyte conditions: 250 mM/20 mM (cis/trans). (B) Conductance of CASTOR at different voltages in presence of potassium. The experiment was done with symmetrical 250 mM KCl buffer in each reservoir (cis/trans). The conductance was recorded at different voltages; crosses represent individual data points. The slope of the linear regression curve (y=0.26x) indicated a maximal conductance of 260 pS.
B A
53
Figure 17. Modelization of the CASTOR pore and the castor-2 mutant pore. (A) Model of the ion pore region of two CASTOR monomers based on the MthK channel (Protein Data Bank code 1LNQ). Amino acid residues constituting the selectivity filter are labeled with the carbonyl oxygen backbone facing the pore. Red: oxygen atoms, blue: amino groups, white: carbon atoms. The alanine 264 (cyan) in CASTOR (B) is substituted with a threonine in castor-2 (C) which potentially results in the formation of two new hydrogen bonds (dashed green lines).
Figure 18. Electrophysiological characterization of castor-2 channel. (A) Selectivity of the castor-2. Voltage ramps (10 mV/s) were applied to bilayers containing four copies of castor-2 at asymmetrical electrolyte conditions: 250 mM/20 mM (cis/trans). (B) Conductance of two copies of castor-2 at different voltages in presence of potassium. The experiment was done with symmetrical 250 mM KCl buffer in each reservoir (cis/trans). The conductance was recorded at different voltages; crosses represent individual data points. The slope of the linear regression curve (y=0.47x) indicated a maximal conductance of 470 pS.
Quaternary ammonium ions (QAs) such as tetrabutylammonium (TBA) or
tetraethylammonium are well established open-K+ channel blockers (Hille, 2001; Yellen,
1984), as well as blocker of the sarcoplasmic reticulum calcium release channels (Tinker
et al., 1992). In contrast, the QAs have no effect on open-cation channels (Demidchik and
Tester, 2002). At a holding potential of 25 mV, the subsequent application of 10, 100,
200 and 500 µM of TBA did not influence the closing of CASTOR or castor-2 inserted in
castor-2
A264T
C CASTOR B
ions
N268 G267 S266
A264 V263
D265
A
y = 0,47x
-60 -40 -20
0 20 40 60
-100 -80 -60 -40 -20 0 20 40 60 80 100 U [mV] U [mV]
I [pA] I [pA]
A B
54
the lipid bilayer (data not shown). All together, the selectivity ratio and the QAs
experiments suggest that CASTOR is a poorly selective cation channel with a better
permeability for potassium.
2.6.2.2 Magnesium mediates voltage-dependent blockage of CASTOR In response to stimuli, ion channels can switch between closed and open state conformations, thereby acting as regulators of ion fluxes in cells. To explore the gating mechanism of CASTOR, we applied different stimuli in order to influence its opening and
closing. Voltage gating was evaluated by modulating the membrane potential from 100
mV to -100 mV in a solution of 250 mM KCl, on both sides of the lipid bilayer. Closing
and opening of CASTOR was observed without any detectable influence of the voltage
(Figure 20A). The current-voltage relation reveals that the channel was active over a
large voltage range from -100 mV to 100 mV without any rectification of the current
(Figure 16B). The current-voltage relationship of CASTOR was linear over this voltage
range with a maximal conductance of 260 pS (Figure 16B). Similarly no rectification of
the current was observed in castor-2 (Figure 18B). A maximal conductance of 235 pS
was observed for castor-2, which is relatively similar to CASTOR.
We tested the influence of divalent cations such as MgCl2 on the gating
mechanism of CASTOR. At positive voltage, random closing steps of CASTOR and
castor-2 were recorded, attesting to the presence of the channels (Figure 19). At these
voltages, no differences were observed between CASTOR and castor-2 in the presence or
absence of 3 mM of MgCl2 on the cis side. However, at negative voltage, and specifically
in the presence of MgCl2, closing of CASTOR occurred at a high frequency, while no
changes were observed for castor-2 (Figure 19). The differences observed between the mutant and the wild type channels demonstrate the specificity of the closing of wild type CASTOR in the presence of Mg2+. This result suggests that the A264T mutation modifies
the pore conformation in such a way that Mg2+ dependent blockage is no longer occurring.
55
Figure 19. Voltage dependent magnesium-blockage of CASTOR. CASTOR and castor-2 channel currents were recorded at voltage indicated on the left, in the presence and absence of physiologically relevant concentration of MgCl2 (3 mM) (Allen and Sanders, 1996). At negative voltage after addition of MgCl2, high frequency of closing steps of CASTOR were observed.
2.6.2.3 CASTOR sensitivity to putative binding ligands
Because InsP3 and Ca2+ are discussed as likely modulators of calcium spiking, we
analysed the sensitivity of CASTOR gating to calcium and InsP3 at a holding potential
from 100 to -100 mV in a recording solution containing 250 mM KCl. The effect of InsP3
has been tested at the maximal physiological concentration of 10 µM (Blatt et al., 1990),
whereas CaCl2 has been applied from 0.1 to 10 mM according to the concentration tested
on MthK (Zadek and Nimigean, 2006). However, neither InsP3 nor Ca2+ influenced
CASTOR gating at the concentrations tested (Figure 20A). A putative NAD(P) binding
domain is predicted at the C-terminus part of CASTOR (Figure 5A). Therefore, NADP+,
NADPH, NAD+ and NADH were tested on the channel gating. An absence of closing
CASTOR -Mg2+ CASTOR +Mg2+ castor-2 -Mg2+ castor-2 +Mg2+
+ 75 mV
+ 50 mV
- 50 mV
- 75 mV
- 100 mV
56
was exclusively observed with 1 mM of NADH, suggesting a binding to CASTOR
(Figure 20B). However, this experiment has been performed only once and further
analyses will be required to get conclusive results.
Figure 20. Modulation of CASTOR gating. (A) Number of closing/opening events at different voltage (control) and after addition of 10 µM inositol triphosphate (+ IP3) or 1 mM calcium chloride (+ CaCl2) during 20s. Standard error of the means of three experiments. (B) Number of closing/opening events at a holding potential of -100 mV after addition of 10 µM IP3, 1 mM CaCl2 or 7 µM NADH during 20s. Standard errors of the means of three experiments for control, IP3 and calcium. Only one experiment has been done with NADH.
Collectively these results demonstrate that either CASTOR or POLLUX can trigger a
flux of potassium and sodium. The electrophysiology experiment highlighted that in the
presence of both cations, CASTOR prefers potassium. Furthermore, the Mg2+ seems to
play a crucial role in the kinetic of CASTOR by reducing its conductance under negative
voltage. In contrast, if the NADH binding is confirmed, this allosteric factor could be
involved in keeping the channel open, possibly in association with another ligand
essential for CASTOR activation.
B A
57
2.7 CASTOR interacting component
The modulation of channel activities by regulatory proteins is a well-known mechanism
in the mammalian system. By biochemical approaches and screening cDNA libraries
using yeast-two-hybrid system, proteins interacting with the C-terminus part of K+ or
cation channels were identified and shown to be involved in modulating current,
promoting the channel assembly or targeting the channel to its active compartment
(Martens et al., 1999; McDonald et al., 1997; Michaelevski et al., 2002; Molokanova et
al., 2000; Scott et al., 1994). Furthermore, mutation in phosphorylation sites combined
with physiological studies provided evidence that K+ channels of the shaker family are
substrates for cAMP-dependent protein kinase (Drain et al., 1994), protein kinase C
(Covarrubias et al., 1994) and Ca2+/calmodulin dependent protein kinase (Roeper et al.,
1997).
In planta, direct interactions between regulatory proteins such as kinase,
phosphatase or calmoduline with K+ or cation channels have been demonstrated via
biochemical or yeast two hybrid approaches (Arazi et al., 2000; Cherel et al., 2002; Xu et
al., 2006). In the early symbiotic signalling pathway, CASTOR and POLLUX are
required for the generation of the calcium spiking as well as the receptor-like kinase,
SYMRK, (Stracke et al., 2002). In tobacco cells, SYMRK:YFP has been localized in the
plasma membrane, as well as in the nucleus (Merixtell Llovera personal communication).
Immunoblot analysis revealed cleavage products of SYMRK which could correspond to
the SYMRK-kinase:YFP fusion relocalized in the nucleus (Merixtell Llovera personal
communication). As a Ca2+ spiking downstream component, a nuclear localized
Ca2+/calmodulin dependent protein kinase (CCaMK) has been identified and is likely to
be a player in deciphering the calcium signature (Levy et al., 2004).
To unravel whether CASTOR activity could be modulated by those symbiotic
components, the yeast two hybrid system was used to check interaction between
CASTOR, SYMRK and CCaMK, as well as to screen a M. loti inoculated-L. japonicus
roots cDNA library.
58
2.7.1 Yeast-two-hybrid assay with common symbiotic components
The C-terminal soluble domains of CASTOR (cCASTOR; H322-E853) and POLLUX
(cPOLLUX; H386-D917) were assayed for interaction with SYMRK-kinase domain and
CCaMK in the yeast two hybrid GAL4 based system. As negative control, the SYMRK
extracellular domain (SYMRK-ECD) and a SYMRK interactor, the Lotus japonicus
seven in absentia 1 (LjSINA1) (Satoko Yoshida personal communication), were used.
However, none of the symbiotic components, SYMRK-kinase domain, CCaMK,
SYMRK-ECD or LjSINA1 were interacted with CASTOR or POLLUX C-terminal
domains (Figure 21).
Figure 21. Yeast-two-hybrid assay for interactions of cCASTOR and cPOLLUX with symbiotic components. Interaction assays were performed in the yeast strain AH109 cotransformed with the bait (BD) and prey (AD) vectors containing either cCASTOR (H322-E853) or cPOLLUX (H386-D917) with SYMRK kinase domain E594-R923 (SYMRK-Kinase), SYMRK extracellular domain T29-Q517 (SYMRK-ECD), CCaMK or LjSINA1. The cotransformants and interacting partners were analysed on synthetic dropout nutrient medium lacking leucine and tryptophan (-LW), and lacking leucine, tryptophan, adenine and histidine (-LWAH), respectively. The constructs BD:SYMRK-Kinase, BD:SYMRK-ECD, BD:LjSNA1 and BD:CCaMK were provided by Satoko Yoshida.
-LW -AHLW
BD:SYMRK-Kinase
BD:SYMRK-ECD
BD:LjSINA1
BD:CCaMK
AD:cCASTOR
BD:cPOLLUX
AD:cPOLLUX
-LW -AHLW
59
2.7.2 Screening of a L. japonicus roots cDNA libraries using yeast-two-hybrid system The C-terminal soluble domain of CASTOR (cCASTOR; H322-E853) was used as a bait
of the yeast two hybrid system to screen a lotus cDNA library constructed in the prey
vector pAD-GAL4-2.1 (Poulsen and Podenphant, 2002). The cDNA library was prepared
from mRNA extracted from L. japonicus seedlings roots 5 and 12 days after M. loti
inoculation (Poulsen and Podenphant, 2002). Approximately 4 million yeast S. cerevisia
colonies expressing the cDNA library were assayed for their ability to grow on selective
synthetic medium (SD/-AHLW). The prey plasmids were isolated from the positive
colonies and cotransformed with the pBD:cCASTOR back to yeast. Colonies that failed
to grow in the second round of testing were considered as false positives. Polymerase
chain reaction (PCR) and restriction digest with RsaI were performed on the positive
clones in order to identify similar sequence pattern. One clone per identical pattern was
sequenced. The 46 sequences obtained were assembled into 7 contigs
(http://staden.sourceforge.net/). BlastX search with the 7 putative interactors identified
similar protein in other legumes as well as in A. thaliana (Table 7). Among them,
proteins similar to an A. thaliana developmental protein DG118 (named 50), a Zinc
finger RING-type protein (named 84) and an A. thaliana pentatrico-peptide (PPR) repeat-
containing protein (named 64) were chosen for further analyses. Both the L. japonicus
sequence-related A. thaliana developmental protein DG118 (50) and the L. japonicus
sequence-related Zinc finger RING-type protein (84) were predicted to localize in the
nucleus (Table 7).
The putative cCASTOR interactors full length 50, 84 and 64 were subcloned and
tested for vice versa interaction, as well as for interaction with cPOLLUX in yeast two
hybrid assays. None of the cCASTOR interactors were found to interact with cPOLLUX
(Figure 22). Moreover, only the candidate 50 interacted with cCASTOR in both
directions reinforcing the hypothesis that 50 is a CASTOR real interactor (Figure 22).
The candidate 50 encodes a small soluble protein of 204 amino acids containing a
predicted Snf7 (sucrose non-fermenting 7) domain which is involved in protein sorting
(Babst et al., 2002; Peck et al., 2004). Therefore, we renamed 50, LjSNF7 for Lotus
japonicus Snf7 proteins.
60
Table 7. List of putative cCASTOR interactors.
Interactor numbers Blast X similarity results Features 50, 55, 51, 123, 100, 61, 70, 108, 66, 112, 18, 19, 22, 01, 34, 21, 35, 12, 29
gi:18410249, A. thaliana vacuolar protein sorting (VPS) 46.2, E value = 1e-29
Pfam03357b, Snf7 domain, involved in protein sorting Localizationa: nucl 6
52, 57, 67, 02, 03, 08, 20, 13, 06
gi:145362649, A. thaliana Eucaryotic translation initiation factor 2 subunit beta (eIF-2-beta), E value = 4e-100
Pfam01873b, contain putative zinc finger binding C domain Localizationa: chlo:4, cyto:4.
65, 120, 64, 04, 05, 17, 32
gi:15233698, A. thaliana pentatrico-peptide (PPR) repeat-containing protein, E value = 2e-65
Pfam01535b, PPR repeat Localizationa: cyto 9.
117, 103, 71, 85, 15
gi:161789859, G. max transcription factor, GT-1, E value = 2e-92
InterProScanb: no hit. Localizationa: nucl 14.
72, 101, 84, 23
gi:124360876, M. truncatula Zinc finger, RING-type, E value = 9e-83
IPR005805b, Rieske iron-sulphur protein domain. Localizationa: nucl 8
114
gi:163889364, M. truncatula bHLH transcription factor, E value = 7e-41
InterProScanb: no hit. Localizationa: chlo 7
24
gi:15231043, A. thaliana chlorophyll synthase, ATG4/ CHLG/G4, E value = 1e-86
Pfam01040b: UbiA Prenyl- transferase Localizationa: nucl 6
a, localization predicted with WolfPSort (http://wolfpsort.org/). nucl, nuclear; chlo; chloroplastic, cyto; cytoplasmic. b, conserved domains predicted with InterProScan (http://www.ebi.ac.uk/Tools/InterPro Scan/). No information concerning the level of expression of the interactors were available in the Lotus japonicus array on the Kazusa website (http://est.kazusa.or.jp/en/plant/lotus/EST/)
61
Figure 22. Yeast-two-hybrid assay for interaction between cCASTOR and yeast two hybrid library full-length candidat interactors. (A) Interaction assays were performed in the yeast strain AH109 cotransformed with the bait (BD) and prey (AD) vectors containing either cCASTOR (H322-E853), cPOLLUX (H386-D917), 84 full length, 50 full length or 64 full length. The cotransformants were analysed on synthetic dropout nutrient medium lacking leucine and tryptophan (-LW). (B) The growth of interacting partners were analysed on synthetic dropout nutrient medium lacking leucine, tryptophan, adenine and histidine (-LWAH).
2.7.2.1 Root nodule phenotype of transformed L. japonicus roots expressing
RNAiLjSNF7 construct.
In order to attest whether LjSNF7 plays a role in root nodule symbiosis, gene silencing
via RNA interference (RNAi) of the interacting candidat was performed in transgenic L.
japonicus roots. A 278 bp sequence corresponding to the 3´ end of the gene was
subcloned under the regulation of the L. japonicus ubiquitin promoter (Ljubq1) in the
pUB-GWS-GFP binary plasmid carrying the GFP fluorescent marker (Maekawa et al.,
2008). As control, the empty vector, as well as the vector carrying a partial sequence of
the β-glucuronidase (GUS) gene, were used (Maekawa et al., 2008). The binary vectors
were transformed in the Agrobacterium rhizogenes AR1193 strain. The transformed
strain was then used to inoculate L. japonicus seedling hypocotyls in order to regenerate
transformed roots. The transgenic roots were scored for nodule formation 3 weeks after
inoculation with the M. loti R7A strain. An average of 2.7 and 2.6 nodules were formed
on the L. japonicus transgenic roots carrying the silencing GUS construct or the empty
vectors, respectively. In contrast, an average of 0.5 nodules appears on the pUB-
GWSLjSNF7-GFP transgenic roots (Figure 23 A). In the pUB-GWSLjSNF7-GFP
cCAS
cPOL AD
empty cCAS
84
64
50
84 64 50
cPOL
A BD
empty
B
cCAS
cPOL AD
empty cCAS
84
64
50
84 64 50
cPOL
BD
empty
62
transgenic roots, the LjSNF7 expression levels were reduced by 50 % in comparison to
the wild type (Figure 23B), which is in line with the 50 % reduction of nodule formation.
Figure 23. Downregulation of LjSNF7 in A. rhizogenes transformed roots expressing pUB-GWSLjSNF7-GFP impaired nodulation. (A) Number of nodules formed after 3 weeks inoculation with the M. loti R7A strain on transgenic roots expressing the silencing cassette LjSNF7 (RNAi50), GUS (RNAiGUS) or the empty cassette (RNAi -) under the regulation of the L. japonicus ubiquitin promoter (Ljubq1). Standard errors of the mean of 23 roots. (B) Relative expression levels of LjSNF7 measured by quantitative RT-PCR in roots expressing pUB-GWSLjSNF7-GFP (RNAi50) or in roots expressing the empty vector pUB-GWS-GFP (RNAi -).
2.7.2.2 Sub-cellular localization of LjSNF7
To determine the sub-cellular localization of LjSNF7, a LjSNF7:RFP fusion construct
was transiently expressed under the regulation of a single P35S promoter in cell culture
derived L. japonicus protoplasts. The fluorescence of the LjSNF7:RFP was compared to
various organelle markers including the plastid marker (FNR:GFP), the endoplasmic
reticulum marker (GFP:HDEL), and as cytoplasmic and nucleoplasmic marker, the free
RFP (Figure 24). The analyses of the LjSNF7:RFP-expressing protoplasts showed that
the red fluorescence was confined in an uncharacterized compartment surrounding the
nucleus which does not correspond to either ER, plastid or a free diffusion of
LjSNF7:RFP (Figure 24M to 24O). In order to determine whether the LjSNF7
fluorescence pattern could correspond to the CASTOR localized compartment, we
generated a fusion construct between the CASTOR N-terminal fragment encompassing
RNAi50 RNAi -
B A
RNAi50 RNAi GUS
RNAi -
63
the first 69 amino acids, including the predicted NLS (Figure 5A), and the RFP. The red
fluorescence generated by the expression of TPcas:RFP was observed in nuclear-
localized patches (Figure 24J to 24L). Although the fluorescence of both fusion
constructs were observed in a nuclear-localized compartment, further experiments
including colocalization, BiFC and co-immunoprecipitation will be required to determine
whether CASTOR and LjSNF7 are really interacting in planta.
Figure 24. Subcellular localization of LjSNF7:RFP in L. japonicus cell culture protoplast. (A) to (C) Epifluorescence microscope pictures of protoplasts expressing the GFP:HDEL fusion proteins (ER control), (D) to (F) the FNR:GFP fusion proteins (plastid control), (G) to (I) RFP (cytosolic and nucleosomal control), (J) to (L) coexpressing FNR:GFP and TPcas:RFP fusion proteins, (M) to (O) the LjSNF7:RFP fusion proteins. (A), (D) and (J) GFP fluorescence; (H), (K) and (N) RFP fluorescence; (B), (E), (G) and (M) bright field images; (C), (F), (I), (L) and (O) Fluorescence and bright field images merged. Bar = 20 µm.
A B C
D E F
G H I
J K L
M N O
64
3. Discussion
Nod factor perception at the plasma membrane triggers perinuclear Ca2+ oscillations
(Ehrhardt et al., 1996). CASTOR and POLLUX are required for Nod factor induced Ca2+
spiking (Miwa et al., 2006). To understand how CASTOR and POLLUX are linked to the
Ca2+ spiking machinery, we investigated the structural and biochemical features of these
two sequence-related proteins.
3.1 Nuclear localization of CASTOR and POLLUX
The immunogold labeling of endogenous CASTOR in L. japonicus clearly demonstrates
the localization of CASTOR in the nuclear envelope. The complementation of a castor
mutant with P35S:POLLUX suggests a similar localization of POLLUX. This result is in
line with the WoLFPSORT analyses, which predict three and one nuclear localization
signals (NLS) in POLLUX and CASTOR, respectively. However, the difference between
CASTOR and POLLUX in number and sequences of predicted NLS suggests that they
may not reside in the same location. The absence of pollux-5 complementation with
P35S:CASTOR could be explained by this possible distinct targeting. In order to reach the
inner nuclear membrane, membrane proteins must move across the three continuous
membrane domains that make up the nuclear envelope: the ONM, the pore membrane
and the INM where they reside. Divers mechanisms have been proposed to explain the
migration of the membrane protein to the INM. Based on study in HeLa cells, one model
suggests that the migration in the pore membrane could be energy dependent (Ohba et al.,
2004). On the other hand, the movement of the integral INM proteins might be promoted
via direct interaction of the membrane protein with specific nucleoporins. An important
feature of the second model would be the recognition of specific signal sequence
similarly to the NLS of soluble nuclear localized proteins. Evidence for the role of NLS
in targeting the inner membrane protein comes from studies in yeast. Mutation or deletion
of NLS of the protein helix-extension-helix-2, Heh2, caused the dispersal of the protein
through the ER (King et al., 2006). Moreover, Heh2 that lacks the NLS were excluded
from the INM (King et al., 2006). The sequence of the NLS identified is enriched in
65
lysine similarly to POLLUX NLS. Although the studies has been done in yeast, the
nucleopore complex machinery seems to be well conserved among plant, fungal and
animal lineages (Bapteste et al., 2005). Therefore, in a hypothetical scenario, given that
the NLS will be membrane tethered, CASTOR resides in the outer nuclear envelope
while POLLUX is required in both the inner and the outer nuclear membrane. Such
partially overlapping localization could explain the absence of pollux-5 complementation
with P35S:CASTOR. However, further experiments will be required to confirm this
hypothesis. Previously, the localization CASTOR and POLLUX, predicted by TargetP to
carry a chloroplast transit peptide, has been reported in plastids of onion and pea root
cells (Imaizumi-Anraku et al., 2005). In this study, we observed plastid localization of
CASTOR and POLLUX GFP fusion constructs in tobacco epidermal cells when driven
by 2xP35S. In the same cellular system, but expressed under the control of 1xP35S,
CASTOR and POLLUX were only observed in the nuclear envelope. Although a
previous report demonstrates that a protein initially targeted to the ER could then be
delivered to the plastid (Villarejo et al., 2005), it is unclear from our study whether
CASTOR and POLLUX are localized to the plastid after being targeted to the secretory
pathway, or directly delivered to the plastid when highly expressed in heterologous cells.
All together, these experiments suggest that CASTOR and POLLUX have the potential to
be dual targeted to the plastid or to the nuclear envelope.
3.2 CASTOR and POLLUX are non-selective cation channels
The functional complementation of a castor mutant by P35S:POLLUX revealed that the
two distinct complexes formed by CASTOR or POLLUX have identical biochemical
function.
In order to characterize the function of CASTOR and its kinetics properties, we
employed electrophysiological assays on CASTOR reconstituted in planar lipid bilayers.
The CASTOR model predicted a decoration of the pore with carbonyl oxygen atoms
which suggests a cation selectivity. With a permeability of approximately 6:1 and 2:1 for
PK/PCl and PNa/PCl, respectively, CASTOR is confirmed as a cation channel weakly
selective for cation over anion. The PCa/PCl of 0.5 demonstrates that CASTOR has a
66
lower permeable for Ca2+ than for Cl-. Considering the oxygen backbone facing the pore
and conferring an electronegative environment, in the presence of Ca2+, a size exclusion
mechanism could explain the chloride flux observed. Although the selectivity of
CASTOR for cation is weak, it appears identical to other endomembrane cation channels
such as the slow and fast vacuolar cation channel of sugarbeet for which a PK/PCl of 6:1
has been recorded (Hedrich and Neher, 1987). In the presence of equimolar concentration
of NaCl or CaCl2, the preference for K+ was confirmed. However, with a 2-fold
preference for K+ over Na+ and Ca2+, CASTOR is only weakly selective for potassium.
Moreover, in line with numerous cation channels characterized (Davenport and Tester,
2000; Demidchik et al., 2002), CASTOR is also insensitive to QAs K+ channel inhibitor.
Furthermore, the high conductance of CASTOR is of the same range as other cation
channels revealed by patch clamp analyses of parsley protoplast (Demidchik et al., 2002;
Zimmermann et al., 1997). Interestingly, a high-conductance cation channel has also been
recorded in beet root nuclear envelope (Grygorczyk and Grygorczyk, 1998).
CASTOR and POLLUX share the same selectivity filter sequences. The
expression of POLLUX in trk1Δ mutant and the complementation of the yeast mutant
strain MAB 2d indicate that POLLUX behaves as a cation channel permeable to both
potassium and sodium.
All together, the electrophysiology and yeast assays indicate unequivocally that
CASTOR and POLLUX are non-selective cation channels permeable to both sodium and
potassium. In the cytoplasm, a low Na+ and high K+ concentration are essential for the
maintenance of a number of enzymatic processes and protein synthesis (Bhandal and
Malik, 1988; Tester and Davenport, 2003). Considering the preference of CASTOR for
potassium, together with the fact that potassium has a concentration as high as 100 to 200
mM within the cytoplasm (Gierth and Mäser, 2007; Walker et al., 1996), we propose that
potassium is likely to be the major ion going through CASTOR and POLLUX in vivo. In
this model, the potassium is supposed to go down a concentration gradient from the
cytoplasm to the perinuclear membrane space.
67
3.3 Channel gating
To explore possible opening and closing mechanisms of CASTOR, we tested effectors
either known to regulate cation channel gating or proposed to be involved in the
signalling pathway leading to Ca2+ spiking. Our results revealed a regulatory mechanism
involving a divalent cation. At negative voltage and in presence of Mg2+, a blockage of
CASTOR conductance was observed. This behaviour is reminiscent of voltage-dependent
divalent cation blockage, a phenomenon which has been studied in detail in potassium
and cation channels (Armstrong et al., 1982; Lu and MacKinnon, 1994; Stern et al., 1987;
Zhang et al., 2006). Different voltage-dependent cation blocking mechanisms have been
observed. A ring of negatively charged amino acids in the conduction pathway formed by
the inner helices is involved in this electrostatic mechanism in a subset of potassium
channels (Zhang et al., 2006). In contrast, in cation channels, the external site of the
selectivity filter can bind divalent cations that serve to block monovalent cation
conduction (Armstrong et al., 1982; Stern et al., 1987). To be active, the voltage-
dependent magnesium blocking mechanism requires a membrane potential positive at the
side of the magnesium source. Cationic lipophilic dyes which accumulate in the organelle
depending on the membrane potential, have been utilized extensively to measure
membrane potential (Marchetti et al., 2004). In living plant cells the cyanine dye 3,3´-
dihexyloxacarbocyanine iodide (DiOC6(3)) (Metivier et al., 1998), was shown to
accumulate in the nuclear envelope suggesting the generation of a membrane potential
across the inner and outer membrane (Matzke and Matzke, 1986). This experiment
suggests that nuclear membrane potential would be negative inside the perinuclear space
relative to the outside cytoplasmic space. In a hypothetical model, the voltage-dependent
magnesium blocking mechanism is active while the nuclear membrane potential
difference reaches a precise negative voltage, attracting the magnesium in the conducting
pathway and limiting the potassium conductance.
The absence of blocking in castor-2 revealed that the A264T mutation modifies
the structure of the pore and filter in such a way that magnesium blockage could no
longer occur. A structural modification of the pore of castor-2 is further supported by the
equal selectivity to K+ and Na+. Our data suggest that the phenomenon of voltage-
dependent Mg2+ blockage also occurs in CASTOR. The absence of this regulatory
68
mechanism in castor-2 could influence potassium fluxes, which may be inappropriate for
calcium spiking.
CASTOR and POLLUX each have large C-terminal domain, which is required for
the homocomplex assembly. In addition, the large C-terminal domain of channels have
been involved in regulating channel conductance via binding of allosteric effectors (Li et
al., 2007). As in M. truncatula, pharmacological studies implicated PLD and PLC
upstream of Ca2+ spiking (den Hartog et al., 2003; Engstrom et al., 2002), phospholipid
derived signaling compounds may be directly or indirectly involved in triggering the Ca2+
oscillation. However, our experiments did not reveal a direct influence of InsP3 or
calcium on CASTOR gating. Nevertheless, an absence of closing of CASTOR in the
presence of NADH suggests a binding of NADH possibly to the predicted NAD binding
domain keeping the channel in an open configuration. However, the experiment was done
at -100 mV in absence of magnesium. Therefore it would be interesting to test whether in
presence of both magnesium and NADH, CASTOR stays in an open configuration.
3.4 Hypothetical role of LjSNF7 in the root nodule symbiosis
A combination of S. cerevisiae genetics and biochemistry has led to the identification of
Snf7 proteins as part of the endosomal sorting complex required for transport of proteins
into the intralumenal vesicles of endosomes (Babst et al., 2002). In this process, Snf7
proteins interact with a vacuolar protein-sorting (Vps20) which bind to the endosomal
membrane in order to form the endosomal vesicular bodies.
Further studies have linked Snf7 to other signalling pathways which involve or
not protein trafficking. Yeast two hybrid analyses and large scale proteomic experiments
demonstrate interaction of Snf7 proteins with multiple proteins including a protease,
Rim13p, and a calpain-like protease, Rim20p (Gavin et al., 2002; Ito et al., 2001).
Rim13p and Rim20p belong to the RIM101 conserved pathway which confer the ability
of fungi to sense and respond to neutral-alkaline environments changes (Davis et al.,
2000b; Li et al., 2004). Rim101p is a transcription factor which promotes changes in gene
expression, acting as inducer of neutral-alkaline response genes and a repressor of acidic
response genes (Bensen et al., 2004; Davis et al., 2000a). Rim20p and Rim13p are
69
proposed to control activation of the transcription factor Rim101p by cleaving it and
releasing the active N-terminal domain which contain the zinc fingers (Xu et al., 2004).
Although the exact function of Snf7p in the RIM101 pathways is unclear, its role as a
scaffold protein bringing the proteases Rim20p and Rim13p to the substrate Rim101p is
suggested (Xu and Mitchell, 2001).
Homologous LjSNF7 proteins are present in numerous monocotyldones and
dicotyledones including A. thaliana (Figure 25). Although putative homologs of all the
main ESCRT-related proteins have been identified in plants (Winter and Hauser, 2006),
only few, excluding the A. thaliana putative homologs, have been functionally studied.
Nevertheless, the roles of SNF7 proteins described have in common the function to
recruit proteins. Therefore, if LjSNF7 protein is a real CASTOR interactor, it potentially
could be involved in the recruitment of protein to CASTOR in order to activate CASTOR
gating or to anchor CASTOR to the outer nuclear membrane.
Figure 25. Unrooted radial phylogenetic tree of LjSNF7 homologs. Homologous full-length protein sequences with E value ≤ 10-68 were aligned using ClustalX and MacClade programs. The protein tree was constructed using the consensus parsimony method (PHYLIP 3.67 program). Percentage bootstrap support based on 1000 replicates is given to the side of each branch. A.t., Arabidopsis thaliana; N.b., Nicotiana benthamiana; V.v., Vitis vinifera; O.s., Oryza sativa; Z.m., Zea mays; H.v., Hordeum vulgare; P.p., Physcomitrella patens. N.b, gi28916459; V.v., gi147855645; A.t.1., gi15220819; A.t.2., gi9802746; A.t.3., gi18410249; A.t.4., gi21537090; Z.m., gi162461720; O.s.1., gi115469156; H.v.1., gi165932189; H.v.2., gi165932187; P.p.1., gi168001188; P.p.2., gi168023705; P.p.3., gi168065046; P.p.4., gi168015068. Bar: number of amino acid substitution per site between two sequence.
A.t.2. A.t.1.
A.t.3. A.t.4. N.b.
V.v.
LjSNF7
P.p.3.
P.p.1.
P.p.4. P.p.2.
Z.m. O.s.1.
H.v.1. H.v.2.
1.1
Dicotyledons
Monocotyledons Moss
72
52
62
52
55 50
50
50
65
50
45
32
37
70
3.5 CASTOR and POLLUX are required for calcium spiking
Because Ca2+ spiking occurs in both the nucleoplasm and the nuclear associated
cytoplasm, the ER and nuclear envelope complex have been proposed to serve as the
corresponding Ca2+ stores (Oldroyd and Downie, 2006). Collectively, our results
demonstrate that CASTOR and POLLUX are cation channels permeable to potassium.
Since potassium contributes significantly to endomembrane potentials, an opening of
CASTOR and POLLUX is likely to influence the potential of the membrane they reside
in. The mutant phenotype and nuclear localization of these channels suggests that they
may be part of the Ca2+ spiking machinery. Furthermore, the high conductance of
CASTOR is similar to that of the potassium channels acting as counter ion channels
balancing the positive charge released from the sarcoplasmic reticulum (Wang and Best,
1994). We therefore propose that CASTOR and POLLUX may act as counter ion
channels facilitating an influx of potassium, which compensates for the rapid release of
positive charge from the calcium store during each spike (Figure 26). A Nod factor-
induced ligand may be involved in CASTOR and POLLUX opening, which would be
temporally coordinated with the yet-to-be-identified Ca2+ channel. To be able to bind the
effector, the putative carboxyterminal regulatory domain would have to be exposed to the
cyto- or nucleoplasm. The predicted direction of ion flow into the nuclear envelope cavity
is consistent with orientation of the pore in such a way that the carboxyterminal portion is
exposed to the outside of the nuclear envelope membranes. Magnesium is an ion present
in millimolar concentrations within the cyto- and nucleoplasm constitutively available
under physiological conditions in the cell (Allen and Sanders, 1996). The Mg2+ blockage
mechanism is likely to be involved in decreasing CASTOR and POLLUX conductance
during spiking, which could lead to a concomitant stop of Ca2+ efflux. The calcium store
would then be replenished by the activity of calcium ATPases. In previous studies,
cyclopiazonic acid, an inhibitor of type IIa calcium-ATPase, inhibited Nod factor induced
Ca2+ spiking (Engstrom et al., 2002), suggesting their involvement in the generation of
Ca2+ spiking.
However, due to the potassium permeability of CASTOR and POLLUX, their
opening could affect the nuclear membrane potential. In mammals, the membrane
potential influences the activation of calcium-induced calcium release channels (Stehno-
71
Bittel et al., 1995). Therefore, CASTOR and POLLUX may, in addition or alternatively
to their role as counter ion channels, be directly involved in regulating electronically
coupled Ca2+ channels on the same membrane system. In M. truncatula, mastoparan was
observed to induce Ca2+ spiking in wild-type as well as in dmi1 mutant root hairs (Sun et
al., 2007). By interrogation of public sequence databases, we identified a putative
CASTOR orthologue of M. truncatula, MtCASTOR (Figure 4). The successful
complementation of castor with POLLUX demonstrates that the twin channels have
similar functions. Moreover, quantitative gene expression levels appear to be critical in
determining the channel mutant phenotypes. It is therefore possible that the mastoparan-
induced Ca2+ spiking in the absence of DMI1 works via an over-activated MtCASTOR
sufficient to bypass the requirement for DMI1.
Figure 26. Proposed role of CASTOR and POLLUX as counter ion channels. During calcium spiking, calcium is released from the nuclear envelope through a yet unidentified calcium channel (white trapezoid). CASTOR and POLLUX facilitate a counter movement of potassium cations to compensate the charge release. A calcium ATPase (white diamond) would then be responsible for actively replenishing the calcium store.
CASTOR and POLLUX are the founder members of a novel cation channel
family. Sequence-related genes to CASTOR and POLLUX are widespread across higher
plants. The presence of members in Arabidopsis thaliana, an asymbiotic species (Figure
4), suggests additional roles of these classes of potassium permeable channels. Ca2+
oscillations are involved in the regulation of different biological process such as pollen
tube growth or guard-cell turgor (Franklin-Tong et al., 1996; McAinsh et al., 1995).
Interestingly, guard cells can integrate information from multiple stimuli such as abscisic
acid, external K+ or Ca2+, to mount appropriate Ca2+ signatures that lead to stomatal
72
closure (Leckie et al., 1998; McAinsh et al., 1995). It will be interesting to determine
whether the A. thaliana channels related to CASTOR and POLLUX are involved in the
generation of Ca2+ oscillations in guard cells.
73
4. Material and methods
4.1 Material
4.1.1 Plants and chemicals
Lotus japonicus ecotype Gifu accession B-129 (Handberg and Stougaard, 1992), L.
japonicus ecotype Gifu accession B-129 EMS mutants (Table 2 and Table 3) and
Nicotiana benthamiana (Wydro et al., 2006) seeds were used in this study.
Chemicals used were purchased from Sigma Aldrich (München, Germany), Roth
(Karlsruhe, Germany), Roche (Penzberd, Germany), Merck (Darmstadt, Germany),
Amersham Bioscience (Freiburg, Germany) and Invitrogen (Karlsruhe, Germany).
4.1.2 Enzymes and kits
The enzymes used in this study were obtained from New England Biolabs (Frankfurt,
Germany), Invitrogen (Karlsruhe, Germany) and MBI Fermentas (St. Leon-Rotl,
Germany). For plasmid DNA extraction the NucleoSpin® Plasmid kit from Macherey-
Nagel (Düren, Germany) was used. For plant RNA extraction the NucleoSpin RNA Plant
kit from Macherey-Nagel (Düren, Germany) was used. For directional cloning strategy
the pENTRTM Directional TOPO® cloning Kits from Invitrogen (Karlsruhe, Germany)
were used. The in vitro transcription-translation was performed with the RTS 100 E. coli
HY and RTS 500 ProteoMaster E. coli HY kits from Roche (Penzberd, Germany).
4.1.3 Strains, oligonucleotides, vectors and clones
The following bacterial and fungal strains have been used in this study: the E.coli TOP10,
DH5α and DB3.1 (Invitrogen), the A. tumefaciens Agl1 (Lazo et al., 1991) and
74
GV3101pMP90RK (Koncz and Schell, 1986), the A. rhizogenes AR1193 (Stougaard et
al., 1987b), the yeast AH109 (Clontech), the yeast mutant MAB 2d (Maresova and
Sychrova, 2005), the cch1Δmid1Δ mutant (Peiter et al., 2005) and trk1Δ mutant (ORF
Y17000 from Euroscarf collection; http://web.uni-frankfurt.de/fb15/mikro/euroscarf/
col_index.html), as well as their respective parental strain JK9-3da (Peiter et al., 2005)
and BY4742 (Euroscarf), the M. loti R7A (Sullivan et al., 1995) and the G. intraradices
BEG195 (http://www.kent.ac.uk/bio/beg/englishhomepage.htm). The oligonucleotides
used were purchased from Metabion (Munich, Germany). The constructs used in this
study are listed in the Table 8A and 8B.
Table 8A. Construct used in this study
Localization of CASTOR and POLLUX Vectors Insert Construct name
pK7FWG2 (Karimi et al., 2002) CASTOR 1x35S:CASTOR:GFP pK7FWG2 (Karimi et al., 2002) POLLUX 1x35S:POLLUX:GFP pK7RWG2 (Karimi et al., 2002) TM1CAS 1x35S:TM1CAS:RFP pK7RWG2 (Karimi et al., 2002) TM1POL 1x35S:TM1POL:RFP pamPAT-MCS (GenBank, AY436765) CASTOR 2x35S:CASTOR:GFP pamPAT-MCS (GenBank, AY436765) POLLUX 2x35S:POLLUX:GFP pK7FWG2 (Karimi et al., 2002)
FNR 1x35S:FNR:GFP
Formation of CASTOR and POLLUX homocomplexes Vectors Insert Construct name
pAD-GAL4-GW cCASTOR AD:cCASTOR pAD-GAL4-GW cPOLLUX AD:cPOLLUX pBD-GAL4-GW cCASTOR BD:cCASTOR pBD-GAL4-GW cPOLLUX BD:cPOLLUX pBD-GAL4-GW ccastor-7 (V598I) BD:ccastor-7 pBD-GAL4-GW ccastor-17 (R590H) BD:ccastor-17 pBD-GAL4-GW ccastor-28 (D537G) BD:ccastor-28 pBD-GAL4-GW ccastor-1 (ΔLA 479-480) BD:ccastor-1 pBD-GAL4-GW ccastor-13 (D444N) BD:ccastor-13 pBD-GAL4-GW cpollux-7 (V531E) BD:cpollux-7 pBD-GAL4-GW cpollux-8 (W781*) BD:cpollux-8 pSPYCE 35S-GW (Walter et al., 2004) CASTOR CASTOR:C pSPYCE 35S-GW (Walter et al., 2004) POLLUX POLLUX:C pSPYNE 35S-GW (Walter et al., 2004) CASTOR CASTOR:N pSPYNE 35S-GW (Walter et al., 2004) POLLUX POLLUX:N pSPYCE 35S-GW (Walter et al., 2004) castor-1 (ΔLA 479-480) castor-1:C pSPYNE 35S-GW (Walter et al., 2004) castor-1 (ΔLA 479-480) castor-1:N pKGWFS7 (Karimi et al., 2002) Promoter CASTOR (2309 bp) PCAS:GFP:GUS pKGWFS7 (Karimi et al., 2002) Promoter POLLUX (2843 bp) PPOL:GFP:GUS pRedRoot_P35S:DMI1:GFP (Riely et al., 2007)
Functional characterization Vectors Insert Construct name
pTMBV4 (Dualsystems Biotech AG) CASTOR pTMBV4:CASTOR pTMBV4 (Dualsystems Biotech AG) POLLUX pTMBV4:POLLUX pDL2xN (Dualsystems Biotech AG) CASTOR pDL2:CASTOR pDL2xN (Dualsystems Biotech AG) POLLUX pDL2:POLLUX pDEST17 (Invitrogen) CASTOR:12His pDEST17:CASTOR pDEST17 (Invitrogen) POLLUX:12His pDEST17:POLLUX pDEST17 (Invitrogen) castor-2 (A264T):12His pDEST17:castor-2
75
Table 8B. Construct used in this study
CASTOR interacting protein Vectors Insert Construct name
pAD-GAL4-GW SNF7 AD:50 pAD-GAL4-GW 84 AD:84 pAD-GAL4-GW 64 AD:64 pBD-GAL4-GW SNF7 BD:50 pBD-GAL4-GW 84 BD:84 pBD-GAL4-GW 64 BD:64 pUB-GWS-GFP (Maekawa et al., 2008) SNF7 pUB-GWS50-GFP pK7RWG2 (Karimi et al., 2002) SNF7 P35S:SNF7:RFP p K7RWG2 (Karimi et al., 2002) TPCAS TPCAS:RFP
4.1.4 Antibodies
The protein expression levels were monitored by immunoblotting using the antibodies
gathered in Table 9.
Table 9. Antibodies used in this study.
Antibodies Dilution Primary antibodies Mouse monoclonal anti-BD (Clontech) 1:1000 Mouse monoclonal anti-c-MYC (Roche) 1:5000 Mouse monoclonal anti-HA (Roche) 1:5000 Rabbit polyclonal anti-CASTOR (Eurogentec) 1:200 Secondary antibodies Dilution IRDye800-conjugated anti-mouse IgG (Biomol) 1:10 000 Alexa Fluor 680 anti-rabbit IgG (Invitrogen) 1:10 000 HRP conjugated anti- rabbit IgG (Amersham) 1: 20 000
76
4.2 Methods
4.2.1 Bioinformatics
Assembly of sequences and mutation detection were performed using the software Staden
Package (http://staden.sourceforge.net/).
CASTOR and POLLUX gene structures from start to stop codons were predicted
using the CODDLE (Codons Optimized to Discovered Deleterious Lesions) program
(http://www.proweb.org./coddle).
To find homologous genes and conserved domains, sequences were analysed by
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), Membrane protein explorer
(http://blanco.biomol.uci.edu/mpex/), EMBL nucleotide sequence database
(http://www.ebi.ac.uk/embl/) and Pfam (http://www.sanger.ac.uk/ software/Pfam/).
Localization signals were predicted by TargetP version 1.01 (http:// www.cbs.dtu.
dk/services/targetP/) and WolfPsort (http://wolfpsort.org/).
Regarding the phylogenetic analyses, ClustalX and MacClade were used to
produce the protein sequence alignment. The consensus parsimony tree was conducted
using PHYLIP 3.67 and displayed using FigTree v1.0. The reliability of the tree was
estimated using the bootstrapping method with 1000 replicates and a random number
generator seed 111.
Sequence-structure homology search was performed using FUGUE v2.0 against a
library of 9673 proteins (Shi et al., 2001). The modelisation was analysed using the
DeepView Swiss-PdbViewer (http://www.expasy.org/spdbv/).
4.2.2 Genetics methods
The map-based cloning of CASTOR was initiated with two populations: an intraspecific
cross between L. japonicus Gifu mutant EMS1749 (castor-2) and L. japonicus
“Miyakojima”, and an interspecific cross between L. japonicus Gifu mutant EMS1749
(castor-2) and L. filicaulis. F2 individuals were previously screened with amplified
77
fragment length polymorphism (AFLP) markers which resulted in located CASTOR at
the south telomeric region of chromosome one (L. Mulder). From one AFLP marker
P21M44 cosegregating with CASTOR, a cleaved amplified polymorphic sequence
(CAPS) marker P21M44/NlaIII was developed and positioned by screening the F2
population to 0.07 cM north of CASTOR on the TAC LjT02K14 or M3 (Figure 2A).
Given that the Lotus genome size is 367 cM (Sandal et al., 2002) or 432 Mb (1 cM = 1.2
Mb), this marker was evaluated at 82 Kb from CASTOR which justified a BAC/TAC
library screening. As no BAC or TAC could be identified or available, a phage library
screening was performed. The Lambda FIXTM IICustom L. japonicus B-129 genomic
library (Stratagene; J. Stougaard) was screened using the P21M44 marker according to
the user manual (http://www.stratagene.com/ manuals/936001.pdf).
4.2.3 Molecular biological methods
4.2.3.1 General molecular biological methods
The general molecular biological methods including bacteria electroporation, bacteria
growth culture, DNA extraction, DNA precipitation, restriction digest, ligation, agarose
gel electrophoresis were performed as described (Sambrook and Russell, 2001). Thereby,
the reaction conditions were adjusted to the recommendations of manufacturers. The
Nanodrop-1000 spectrophotometer (www.nanodrop.com) was used to determine DNA
and RNA amount.
4.2.3.2 TILLING
TILLING was carried out using the general TILLING population and the pre-selected
nodule mutant population described previously (Perry et al., 2003). Primers amplifying a
1-1.5 kb region were designed using the CODDLE program to identify the region that
had the maximum likelihood of being affected by EMS mutation to produce a deleterious
allele. CASTOR and POLLUX-specific forward and reverse primers were directly labelled
with the fluorescent dyes 6-carboxyfluorescein and 4,7,2´,7´-tetrachloro-6-
carboxyfluorescein, respectively, for analysis with an ABI377 sequencer as previously
described (Perry et al., 2003).
78
Genomic DNA isolated from the two populations was diluted to 5ng/µL.
Normalized DNA was pooled 4-fold and 1 µL pooled DNA was used in 10 µL PCR
reaction using the labelled primers as described (Colbert et al., 2001), but without the
addition of unlabeled primers. The touchdown PCR program used was; 95°C 2 min, 7 x
[94°C 20s, Tm + 3°C to Tm – 4°C for 30s, -1°C per cycle, gradient to 72°C at 0.5°C/s,
72°C 1 min], 44 x [94°C 20s, Tm-5°C 30s, gradient to 72°C at 0.5°C/s, 72°C 1 min], 69 x
[72°C 5 min, 99°C 10 min, 70°C for 20s, -0.3°C per cycle], 15 °C for ever, with an
annealing temperature (Tm) of 65°C.
4.2.3.3 Cloning strategies
PCR fragments containing a 3´-A overhangs were cloned into the pGEM®-T Easy vector
(Promega) according to the user manual (www.promega.com). For expression in
eukaryotic system, cDNA were excised from the pGEM®-T Easy vector with compatible
restriction enzyme and subcloned as described (Sambrook and Russell, 2001).
PCR fragments containing a 5´-CACC overhang were cloned into the Gateway
entry vectors pENTR/SD/D-TOPO® or pENTR/D-TOPO® (Invitrogen), and
subsequently subcloned into destination expression vectors for prokaryotic and
eukaryotic expression according to the user manual (http://www.invitrogen.com/).
4.2.3.4 RNA isolation
RNA isolation from L. japonicus roots was performed according to the user manual
(http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/RNA%20and
%20mRNA/UM_TotalRNAPlant.pdf).
4.2.3.5 Reverse transcription (RT)-PCR, Rapid Amplification of cDNA ends
(RACE)-PCR and quantitative Reverse Transcription (qRT)-PCR
The RT- and RACE-PCR were done following the user manual of SMARTTM RACE
cDNA Amplification from Clontech (http://www.genex.cl/stock/clon634914.html),
whereas the qRT-PCR was performed with 100 ng of RNA following the instruction of
the manufacturer, Invitrogen (http://tools.invitrogen.com/content/sfs/manuals/superscript
2 step_qrtpcr_sybr_man.pdf).
79
4.2.3.6 Site directed mutagenesis
The mutations were generated by site directed mutagenesis as described (Horton et al.,
1989).
4.2.3.7 Yeast transformation methods
Yeast two hybrid library screening and interaction analyses were performed in the yeast
strain AH109 according to the user manual (Yeast Protocols Handbook, PT3024-1,
Clontech).
AH109, trk1Δ, BY4742, cch1Δmid1Δ, JK9-3da and MAB 2d yeast strains were
transformed using the lithium acetate method (Gietz and Woods, 2001). Transformants
were screened on SD medium containing 0.67% yeast nitrogen base, 2% glucose, and
lacking appropriate nutrients.
4.2.4 Biochemical methods
4.2.4.1 General biochemical methods
The protein concentration was determine by Bradford assay (Bradford, 1976) using the
Bio-rad protein assay dye reagent concentrate according to the manufacturer manual
(http://www.technomedica.com/publikazii/belur/Bio-Rad.pdf). Precipitation of protein by
acetone or trichloroacetic acid, SDS-PAGE and gel staining by Coomassie Brilliant Blue
R250, were done according to published procedures (Sambrook and Russell, 2001). For
immunodetection proteins were transferred on polyvinylidene fluoride (PVDF)
membrane (Amersham) by electroblotting. Immunodecoration and detection either by
using the alkaline phophatase system or infrared fluorescence were performed according
to (Sambrook and Russell, 2001).
4.2.4.2 Protein extraction from L. japonicus root
In order to get the total protein extract from L. japonicus roots, 3-week-old roots were
ground with liquid nitrogen, subsequently mixed with extraction buffer (100 mM Tris-
HCl pH 7.2, 150 mM NaCl, 5 mM EDTA, 10 mM DTT, 5% SDS, 4 M Urea) and boiled
80
10 min. Debris were precipitated before adding 2xSDS sample buffer and loading 10%
SDS-PAGE.
4.2.4.3 Protein extraction from N. benthamiana leaves
Total crude protein from 2 cm2 of transformed leaves was extracted as described (Walter
et al., 2004).
4.2.4.4 Protein extraction from S. cerevisiae
The crude yeast protein extraction was performed as described in the user manual
(DUALmembrane kit 3, P01001, Dualsystems Biotech) with the following modifications;
50 mL of selective media were inoculated with 500 µL of preculture at 30°C until OD600
reaches 0.6.
4.2.4.5 In vitro expression and purification
CASTOR, castor-2 and POLLUX coding sequence were subcloned into the pDEST17
vector (Invitrogen). To test the expression of CASTOR and POLLUX in a cell free
system, a rapid expression screen was performed with the RTS 100 E. coli HY kit
(Roche) according to the manufacturer instructions. To scale up the protein amount,
CASTOR and castor-2 were expressed using the RTS 500 ProteoMaster E. coli HY Kit
(Roche) according to the manufacturer instructions. The solubilization and purification of
proteins were performed by selective solubilization. The pellets of the cell-free reaction
containing CASTOR or castor-2 were washed twice in 1 mL of washing buffer (100 mM
NaH2PO4, 10 mM Tris, pH 7.2) and centrifuged for 5 min at 5000 g. To remove E. coli
proteins, the pellet was suspended in E. coli protein solubilization buffer (100 mM
NaH2PO4, 2% MEGA-9, 10 mM Tris, pH 7.2), 45 min at 40°C under shaking and
centrifuged for 30 min at 50,000 g. This step was repeated twice. Protein was solubilized
in 100 mM NaH2PO4, 0,05 % SDS, 10 mM Tris, pH 7.2) for 2 h at room temperature
with shaking. Insoluble proteins were precipitated by centrifugation for 30 min at 50,000
g.
81
4.2.5 Electrophysiological methods
Purified CASTOR proteins were dialysed against 100 mM NaH2PO4, 10 mM Tris, 8 M
urea pH 7.2 for 2h at room temperature. To remove any remaining SDS, the dialysed
proteins were incubated with Serdolit® PAD I beads (SERVA) for 3 hours. The protein
concentrations were determined by Bradford assays (Biorad).
Figure 27. Representation of the fusion of proteoliposome with the planar lipids bilayer.
Figure 28. Lipid-bilayer-measurement system. (A) Representation schematic of the system. An ion channel is inserted in a lipid bilayer which split the cis and trans chambers. In each chamber, ion solution is exchanged by perfusion (B) Electrical view of the system. A; Amperemeter, Ucom; voltage generator, CM; lipid bilayer as a capacitor, Rc; canal protein as a resistor.
The reconstitution of proteoliposomes and electrophysiological measurements were
performed according to (Ertel et al., 2005) with the following modification: after
H2O
cis trans
[K+Cl-] [K+Cl-]
cis trans
[K+Cl-] [K+Cl-]
cis trans
[K+Cl-] [K+Cl-]
A B C
B
Rc
CM
A
Ucom
Headstage Measuring amplifier A/D-converter PC
A
cis trans
82
formation of a stable lipid bilayer, the solution of the cis chamber was exchanged with
250 mM KCl, 10 mM MOPS/Tris, pH 7.0. After fusion of the proteoliposome to the
planar lipid bilayer (Figure 27), the recording of the reverse potential was done in a 10
mM MOPS/Tris, pH 7.0 solutions containing in the cis/trans chambers (Figure 28) either
250 mM/20 mM KCl, 250 mM/20 mM NaCl or 125 mM/10 mM CaCl2. The conductance
and current properties were determined in equal solutions of 250 mM KCl, 10 mM
MOPS/Tris, pH 7.0. The background noise of the bilayer was around 8 pS.
4.2.6 Plant transformation methods
4.2.6.1 A. tumefaciens mediated transient transformation
BiFC and localization of the GFP fusion proteins were performed in 5-week-old
Nicotiana benthamiana plants following Agrobacterium tumefaciens GV3101pMP90RK
(Koncz and Schell, 1986) and Agl1 strain-mediated transient transformation, respectively,
according to (Lazo et al., 1991; Walter et al., 2004).
4.2.6.2 A. rhizogenes-mediated transient transformation
The A. rhizogenes strain AR1193 (Stougaard et al., 1987a) was used to transform Lotus
japonicus (ecotype Gifu) wild type and mutant. Seeds were sterilised as previously
described (Hansen et al., 1989) and incubated at 4°C overnight, followed by germination
on B5 medium (Gamborg et al., 1968) in a growth chamber (3 days dark, 3 days short
light, 70% humidity, 24°C). The emerging roots were cut and hypocotyls dipped in
AR1193 carrying the relevant plasmids (OD600=1). Transformed plants were grown on
B5 medium (containing 40 mg/mL kanamycin for plants transformed with the BiFC
construct) in a growth chamber (2 days dark/3 days short light), and then transferred to
B5 medium containing cefotaxim (300 mg/mL). Shoots with developed hairy roots were
dipped into a dilution of a Mesorhizobium loti strain R7A culture (OD600=0.01) and
transplanted into sterile pots containing 300 mL of seramis (Seramis®) and 100 mL
Fahraeus medium (Fahraeus, 1957). Regarding the silencing experiments, the
untransformed roots were cut before inoculation with M. loti R7A strain to avoid
autoregulatory inhibition of nodule formation (Caetano-Anolles and Gresshoff, 1991).
83
4.2.7 Cell biological methods
4.2.7.1 L. japonicus cell culture protoplast transfection
L. japonicus protoplasts were isolated from 5-day-old cell culture. 50 mL of cell culture
was harvested at 860 g, 3 min, washed in 25 mL 240 mM CaCl2 solution and
subsequently incubated in enzyme solution containing 3.5% Cellulase (Onozuka R10),
0.6% Macerozyme (Serva R-10) in 240 mM CaCl2, 14h, 24°C, under 60 rpm shaking.
The proptoplasts were filtered through 40 µm nylon mesh, precipitated 3 min at 860 g,
and washed with 240 mM CaCl2. The pellet was resuspended in 25 mL P5 medium
(1xGamborg B5 medium, 283 mM sucrose, 4,5 µM 2,4 Dichlorophenoxyacetic acid, pH
5.7 with NaOH). The protoplast solution was centrifuged at 300 g 5 min. The floating
protoplasts were harvested and diluted in 15 mL P5 medium to be centrifuged 5 min at
300 g.
The density of viable protoplasts was evaluated by counting the protoplast stained
with 0.24 µM Fluorescein diacetate in a Mallassez counting chamber The following
calculation was applied: if (a) represents the dilution of the sample and (n) the total
number of cells in a unit sample with a volume of (b), then, the load, expressed in
millions of cells per mL, can be calculated by using the equation: millions of cells/mL = a
x n / b x 4.
A minimum amount of 106 protoplasts in a 200 µL volume were transfected as
follows: in a 2 mL eppendorf tubes 10 to 30 µg DNA, 10 µg salmon sperm single
stranded DNA, 200 µL protoplasts, 200 µL polyethylene glycol (PEG) solution (25%
PEG6000, 100 mM Ca(NO3)2, 450 mM mannitol, pH 9.0 with KOH) were added in this
order before shaking. The reaction was incubated 30 min. The protoplasts were
precipitated with 1.5 mL 275 mM Ca(NO3)2 30s at 860 g, to be finally resuspended in 0.5
mL P5 medium. The results were checked after 24h incubation at 24°C.
84
4.2.8 Histochemical methods
4.2.8.2 Immunogold electron microscopy
CASTOR and POLLUX peptides antibodies were raised in rabbits against the peptides:
H2N-SSPSQYGRRFHTNSNT-CONH2 and H2N-CRRGSLPKDFVYPKSP-CONH2 for
CASTOR, and H2N-TTRKRRPSSVKPPST-CONH2 and H2N-GFFPRIPDAPKYPEK-
CONH2 for POLLUX. One-week-old seedlings were used for immunogold electron
microscopy. Pieces of roots were fixed immediately after collection with 2.5%
formaldehyde in 75 mM sodium cacodylate, 2 mM MgCl2, pH 7.0 for 2 h at room
temperature. The tissue was washed 4 times at room temperature with 1% glycine, 2.5%
formaldehyde in 75 mM sodium cacodylate, 2 mM MgCl2, pH 7.0. After two washing
steps in distilled water, the tissue pieces were dehydrated with a graded series of acetone
(20-100%). Tissue samples were then infiltrated and embedded in Spurr’s low-viscosity
resin. After polymerisation, sections with a thickness of 70-90 nm were cut with a
diamond knife and mounted on collodion-coated nickel grids. Ultra-thin sections were
incubated first in 50 mM glycine in PBS buffer (136 mM NaCl, 2 mM KCl, 4 mM
Na2HPO4, 1 mM KH2PO4, pH 7.4) for 20 min, then in blocking buffer (0.5% BSA,
0.01% Tween 20, 0.05% gelatine, in PBS buffer) for 1 h. Sections were incubated with
purified anti-CASTOR serum (Eurogentec) in blocking buffer (diluted 1:20 to 1:100)
overnight at 4°C. After six washing steps with blocking buffer, anti-rabbit-IgG
conjugated with 5 nm gold (Sigma) was added at a dilution of 1:20 (with blocking buffer)
and incubated for 90 min at room temperature. After three washings with blocking buffer,
1% glycine in PBS buffer and distilled water, sections were post stained with aqueous
lead citrate. The sections were used either unstained or post stained with aqueous lead
citrate (100 mM, pH 13.0). Micrographs were taken with a Zeiss EM 912 electron
microscope equipped with an integrated OMEGA energy filter operated in the zero loss
mode. Gold particles were counted on 15 micrographs (magnification 40000x) as
described (Lucocq, 1994). For the nuclear envelope, the “covered area” was a 5 mm wide
strip around the nucleus. The surface of each organelle was calculated and normalized as
percentage of the cell. The density of gold particles was calculated by dividing the
number of gold particles in each compartment by their normalized surface (in
percentage).
85
4.2.8.2 GUS assay
To performed the GUS staining, transformed seedlings were incubated 40 min at 37 °C
with 2 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid, 50 mM NaPO4 pH 7, 1 mM
EDTA, 0,1 % Triton X-100 and subsequently washed in 70 % ethanol.
4.2.8.3 Mycorrhiza staining
Root fragments 2 cm long were incubated in 0.8 mL, 96-well plate (ABgene House). The
staining was performed with black ink according to (Kosuta et al., 2005).
4.2.9 Fungal and bacterial inoculation methods
4.2.9.1 Inoculation with G. intraradices
The Glomus intraradices infection has been performed in the chive nurse pot system.
Two young chive plants preliminarily infected by G. intraradices according to (Kosuta et
al., 2005), are grown in pots of 8.5 cm3 containing substrate composed of a 1:1 v/v
mixture of vermiculite and Quartz sand. After two to six weeks, L. japonicus 2-week-old
seedlings are transferred or scarified non-germinated seeds are sown directly in the nurse
chive pots. Once a week the chive nurses are watered with nutrient solution (24.65 mg/L
MgSO4-7H2O, 5.05 mg/L KNO3, 0.115 mg/L KH2PO4, 0.725 mg/L K2HPO4, 18.55 mg/L
CaCl2.2H2O, 0.0715 mg/L H3BO3, 0.051 mg/L MnSO4.4H2O, 0.011 mg/L ZnSO4.7H2O,
0.004 mg/L CuSO4.5H2O, 0.0025 mg/L Na2MoO4.5H2O, 0.005 mg/L CoCl2.4H2O, 0.835
mg/L Fe-EDTA-di-hydroxy-phenylacetate). The development of arbuscules is checked
after 3 weeks.
4.2.9.2 Inoculation with M. loti
M. loti strain R7A colony is grown two days at 30°C in TY medium (5 g/L Bacto
triptone, 5 g/L Yeast extract, 6 mM CaCl2). The bacteria are pelleted by centrifugation at
8000 rpm 2 min, washed in saline (0.8% NaCl) 2 times, and resuspended at OD600 = 0.01
before inoculation.
86
5. References
Akiyama, K., Matsuzaki, K. and Hayashi, H. (2005) Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature, 435, 824-827.
Albrecht, C., Geurts, R. and Bisseling, T. (1999) Legume nodulation and mycorrhizae formation; two extremes in host specificity meet. Embo J, 18, 281-288.
Alexander, T., Meier, R., Toth, R. and Weber, H.C. (1988) Dynamics of arbuscule development and degenration in mycorrhizas of Triticum aestivum L. and Avena sativa L. with reference to Zea mays L. New Phytologist, 110, 363-370.
Allen, G.J. and Sanders, D. (1996) Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium. Plant J, 10, 1055-1069.
Ane, J.M., Kiss, G.B., Riely, B.K., Penmetsa, R.V., Oldroyd, G.E., Ayax, C., Levy, J., Debelle, F., Baek, J.M., Kalo, P., Rosenberg, C., Roe, B.A., Long, S.R., Denarie, J. and Cook, D.R. (2004) Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science, 303, 1364-1367.
Arazi, T., Kaplan, B. and Fromm, H. (2000) A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains. Plant Mol Biol, 42, 591-601.
Ardourel, M., Demont, N., Debelle, F., Maillet, F., de Billy, F., Prome, J.C., Denarie, J. and Truchet, G. (1994) Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses. Plant Cell, 6, 1357-1374.
Armstrong, C.M., Swenson, R.P., Jr. and Taylor, S.R. (1982) Block of squid axon K channels by internally and externally applied barium ions. J Gen Physiol, 80, 663-682.
Babst, M., Katzmann, D.J., Estepa-Sabal, E.J., Meerloo, T. and Emr, S.D. (2002) Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev Cell, 3, 271-282.
Bapteste, E., Charlebois, R.L., MacLeod, D. and Brochier, C. (2005) The two tempos of nuclear pore complex evolution: highly adapting proteins in an ancient frozen structure. Genome Biol, 6, R85.
Bensen, E.S., Martin, S.J., Li, M., Berman, J. and Davis, D.A. (2004) Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p. Mol Microbiol, 54, 1335-1351.
Besserer, A., Puech-Pages, V., Kiefer, P., Gomez-Roldan, V., Jauneau, A., Roy, S., Portais, J.C., Roux, C., Becard, G. and Sejalon-Delmas, N. (2006) Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria. PLoS Biol, 4, e226.
Bezanilla, F. (2000) The voltage sensor in voltage-dependent ion channels. Physiol Rev, 80, 555-592.
Bhandal, I.S. and Malik, C.P. (1988) Potassium estimation, uptake, and its role in the physiology and metabolism of flowering plants. International Review of Cytology, 110, 205-254.
Blatt, M.R., Thiel, G. and Trentham, D.R. (1990) Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate. Nature, 346, 766-769.
87
Bonfante, P., Genre, A., Faccio, A., Martini, I., Schauser, L., Stougaard, J., Webb, J. and Parniske, M. (2000) The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells. Mol Plant Microbe Interact, 13, 1109-1120.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-254.
Buee, M., Rossignol, M., Jauneau, A., Ranjeva, R. and Becard, G. (2000) The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates. Mol Plant Microbe Interact, 13, 693-698.
Caetano-Anolles, G. and Gresshoff, P.M. (1991) Plant genetic control of nodulation. Annu Rev Microbiol, 45, 345-382.
Calero, F., Gomez, N., Arino, J. and Ramos, J. (2000) Trk1 and Trk2 define the major K(+) transport system in fission yeast. J Bacteriol, 182, 394-399.
Cardenas, L., Dominguez, J., Quinto, C., Lopez-Lara, I.M., Lugtenberg, B.J., Spaink, H.P., Rademaker, G.J., Haverkamp, J. and Thomas-Oates, J.E. (1995) Isolation, chemical structures and biological activity of the lipo-chitin oligosaccharide nodulation signals from Rhizobium etli. Plant Mol Biol, 29, 453-464.
Cardenas, L., Feijo, J.A., Kunkel, J.G., Sanchez, F., Holdaway-Clarke, T., Hepler, P.K. and Quinto, C. (1999) Rhizobium nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs. Plant J, 19, 347-352.
Cardenas, L., Vidali, L., Dominguez, J., Perez, H., Sanchez, F., Helper, P.K. and Quinto, C. (1998) Rearrangement of actin microfilaments in plant root hairs responding to Rhizobium etli nodulation signals. Plant Physiology, 116, 871-877.
Carlson, R.W., Forsberg, L.S., Price, N.P., Bhat, U.R., Kelly, T.M. and Raetz, C.R. (1995) The structure and biosynthesis of Rhizobium leguminosarum lipid A. Prog Clin Biol Res, 392, 25-31.
Catoira, R., Galera, C., de Billy, F., Penmetsa, R.V., Journet, E.P., Maillet, F., Rosenberg, C., Cook, D., Gough, C. and Denarie, J. (2000) Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. Plant Cell, 12, 1647-1666.
Catoira, R., Timmers, A.C., Maillet, F., Galera, C., Penmetsa, R.V., Cook, D., Denarie, J. and Gough, C. (2001) The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling. Development, 128, 1507-1518.
Chabaud, M., Vénard, C., Defaux-Petras, A., Bécard, G. and Barker, D.G. (2002) Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi. New Phytologist, 156, 265-273.
Charron, D., Pingret, J.L., Chabaud, M., Journet, E.P. and Barker, D.G. (2004) Pharmacological evidence that multiple phospholipid signaling pathways link Rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression. Plant Physiol, 136, 3582-3593.
Cherel, I., Michard, E., Platet, N., Mouline, K., Alcon, C., Sentenac, H. and Thibaud, J.B. (2002) Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Plant Cell, 14, 1133-1146.
88
Christie, M.J. (1995) Molecular and functional diversity of K+ channels. Clin Exp Pharmacol Physiol, 22, 944-951.
Colbert, T., Till, B.J., Tompa, R., Reynolds, S., Steine, M.N., Yeung, A.T., McCallum, C.M., Comai, L. and Henikoff, S. (2001) High-throughput screening for induced point mutations. Plant Physiol, 126, 480-484.
Covarrubias, M., Wei, A., Salkoff, L. and Vyas, T.B. (1994) Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate. Neuron, 13, 1403-1412.
Davenport, R.J. and Tester, M. (2000) A weakly voltage-dependent, nonselective cation channel mediates toxic sodium influx in wheat. Plant Physiol, 122, 823-834.
Davis, D., Edwards, J.E., Jr., Mitchell, A.P. and Ibrahim, A.S. (2000a) Candida albicans RIM101 pH response pathway is required for host-pathogen interactions. Infect Immun, 68, 5953-5959.
Davis, D., Wilson, R.B. and Mitchell, A.P. (2000b) RIM101-dependent and-independent pathways govern pH responses in Candida albicans. Mol Cell Biol, 20, 971-978.
de Ruijter, N.C.A., Rook, M.B., Bisseling, T. and Emons, A.M.C. (1998) Lipochito-oligosacharides re-initiate root hair tip growth in Vicia sativa with high calcium and spectrin-like antigen at the tip. Plant Journal, 13, 341-350.
Demidchik, V., Davenport, R.J. and Tester, M. (2002) Nonselective cation channels in plants. Annu Rev Plant Biol, 53, 67-107.
Demidchik, V. and Tester, M. (2002) Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots. Plant Physiol, 128, 379-387.
den Hartog, M., Musgrave, A. and Munnik, T. (2001) Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation. Plant J, 25, 55-65.
den Hartog, M., Verhoef, N. and Munnik, T. (2003) Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells. Plant Physiol, 132, 311-317.
Doyle, J.J. (1998) Phylogenetic perspectives on nodulation: evolving views of plants and symbiotic bacteria. Trends Plant Science, 3, 473-478.
Drain, P., Dubin, A.E. and Aldrich, R.W. (1994) Regulation of Shaker K+ channel inactivation gating by the cAMP-dependent protein kinase. Neuron, 12, 1097-1109.
Drissner, D., Kunze, G., Callewaert, N., Gehrig, P., Tamasloukht, M., Boller, T., Felix, G., Amrhein, N. and Bucher, M. (2007) Lyso-phosphatidylcholine is a signal in the arbuscular mycorrhizal symbiosis. Science, 318, 265-268.
Ehrhardt, D.W., Atkinson, E.M. and Long, S.R. (1992) Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors. Science, 256, 998-1000.
Ehrhardt, D.W., Wais, R. and Long, S.R. (1996) Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell, 85, 673-681.
Endre, G., Kereszt, A., Kevei, Z., Mihacea, S., Kalo, P. and Kiss, G.B. (2002) A receptor kinase gene regulating symbiotic nodule development. Nature, 417, 962-966.
Engstrom, E.M., Ehrhardt, D.W., Mitra, R.M. and Long, S.R. (2002) Pharmacological analysis of nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of type IIA calcium pumps and phosphoinositide signaling. Plant Physiol, 128, 1390-1401.
89
Ertel, F., Mirus, O., Bredemeier, R., Moslavac, S., Becker, T. and Schleiff, E. (2005) The evolutionarily related beta-barrel polypeptide transporters from Pisum sativum and Nostoc PCC7120 contain two distinct functional domains. J Biol Chem, 280, 28281-28289.
Esseling, J.J., Lhuissier, F.G. and Emons, A.M. (2003) Nod factor-induced root hair curling: continuous polar growth towards the point of nod factor application. Plant Physiol, 132, 1982-1988.
Fahraeus, G. (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol, 16, 374-381.
Felle, H.H., Kondorosi, E., Kondorosi, A. and Schultze, M. (1995) Nod signal-induced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications of the lipochitooligosaccharide. Plant Journal, 7, 939-947.
Felle, H.H., Kondorosi, E., Kondorosi, A. and Schultze, M. (1996) Rapid alkalinization in alfalfa root hairs in response to rhizobial lipochitooligosaccharide signals. Plant Journal, 10, 295-301.
Felle, H.H., Kondorosi, E., Kondorosi, A. and Schultze, M. (1998) The role of ion fluxes in Nod factor signalling in Medicago sativa. Plant Journal, 13, 455-463.
Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon, G. and Sanders, D. (1997) The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating. FEBS Lett, 419, 259-262.
Fisher, R.F. and Long, S.R. (1992) Rhizobium--plant signal exchange. Nature, 357, 655-660.
Franklin-Tong, V.E., Drobak, B.K., Allan, A.C., Watkins, P. and Trewavas, A.J. (1996) Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate. Plant Cell, 8, 1305-1321.
Fukami, K. (2002) Structure, regulation, and function of phospholipase C isozymes. J Biochem, 131, 293-299.
Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiba, K. (1989) Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature, 342, 32-38.
Gaber, R.F., Styles, C.A. and Fink, G.R. (1988) TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol Cell Biol, 8, 2848-2859.
Gamborg, O.L., Miller, R.A. and Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res, 50, 151-158.
Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M.A., Copley, R.R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G. and Superti-Furga, G. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415, 141-147.
Genre, A. and Bonfante, P. (2007) Dissection of plant cell responses to arbuscular mycorrhizal fungi reveals an unpredicted role for DMI3, a calcium/calmodulin-
90
dependent kinase. XIII International Congress on Molecular Plant-Microbe Interactions, Sorrento, Italy, p. 188.
Genre, A., Chabaud, M., Faccio, A., Barker, D.G. and Bonfante, P. (2008) Prepenetration Apparatus Assembly Precedes and Predicts the Colonization Patterns of Arbuscular Mycorrhizal Fungi within the Root Cortex of Both Medicago truncatula and Daucus carota. Plant Cell.
Genre, A., Chabaud, M., Timmers, T., Bonfante, P. and Barker, D.G. (2005) Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection. Plant Cell, 17, 3489-3499.
Gerasimenko, O. and Gerasimenko, J. (2004) New aspects of nuclear calcium signalling. J Cell Sci, 117, 3087-3094.
Gianinazzi-Pearson, V. (1996) Plant Cell Responses to Arbuscular Mycorrhizal Fungi: Getting to the Roots of the Symbiosis. Plant Cell, 8, 1871-1883.
Gianinazzi-Pearson, V., Arnould, C., Oufattole, M., Arango, M. and Gianinazzi, S. (2000) Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco. Planta, 211, 609-613.
Gierth, M. and Mäser, P. (2007) Potassium transporters in plants - Invlovement in K+ acquisition, redistribution and homeostasis. FEBS Letters, 581, 2348-2356.
Gietz, R.D. and Woods, R.A. (2001) Genetic transformation of yeast. Biotechniques, 30, 816-820, 822-816, 828 passim.
Gleason, C., Chaudhuri, S., Yang, T., Munoz, A., Poovaiah, B.W. and Oldroyd, G.E. (2006) Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition. Nature, 441, 1149-1152.
Godfroy, O., Debelle, F., Timmers, T. and Rosenberg, C. (2006) A rice calcium- and calmodulin-dependent protein kinase restores nodulation to a legume mutant. Mol Plant Microbe Interact, 19, 495-501.
Goldman, D.E. (1943) Potential, impedance, and rectification in membranes. J. Gen. Physiol., 27, 37-60.
Govindarajulu, M., Pfeffer, P.E., Jin, H., Abubaker, J., Douds, D.D., Allen, J.W., Bucking, H., Lammers, P.J. and Shachar-Hill, Y. (2005) Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature, 435, 819-823.
Grygorczyk, C. and Grygorczyk, R. (1998) A Ca2+- and voltage-dependent cation channel in the nuclear envelope of red beet. Biochim Biophys Acta, 1375, 117-130.
Handberg, K. and Stougaard, J. (1992) Lotus japonicus, an autogamous, diploid legume species fro classical and molecular genetics. Plant Journal, 2, 487-496.
Hansen, J., Jorgensen, J.-E., Stougaard, J. and Marker, K. (1989) Hairy roots - a shortcut to transgenic root nodules. Plant Cell Reports, 8, 12-15.
Harris, J.M., Wais, R. and Long, S.R. (2003) Rhizobium-lnduced calcium spiking in Lotus japonicus. Mol Plant Microbe Interact, 16, 335-341.
Harrison, M.J. (1996) A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations. Plant J, 9, 491-503.
Harrison, M.J., Dewbre, G.R. and Liu, J. (2002) A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi. Plant Cell, 14, 2413-2429.
Hayashi, M., Miyahara, A., Sato, S., Kato, T., Yoshikawa, M., Taketa, M., Hayashi, M., Pedrosa, A., Onda, R., Imaizumi-Anraku, H., Bachmair, A., Sandal, N.,
91
Stougaard, J., Murooka, Y., Tabata, S., Kawasaki, S., Kawaguchi, M. and Harada, K. (2001) Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population. DNA Res, 8, 301-310.
Hedrich, R. and Neher, E. (1987) Cytoplasmic calcium regulates voltage-dependent ion channels in plant vacuoles. Nature, 329, 833-836.
Heginbotham, L., Lu, Z., Abramson, T. and MacKinnon, R. (1994) Mutations in the K+ channel signature sequence. Biophys J, 66, 1061-1067.
Heidstra, R., Geurts, R., Franssen, H., Spaink, H.P., Van Kammen, A. and Bisseling, T. (1994) Root Hair Deformation Activity of Nodulation Factors and Their Fate on Vicia sativa. Plant Physiol, 105, 787-797.
Herendeen, P.S., Magallón-Puebla, S., Lupia, R., Crane, P.R. and Kobylinska, J. (1999) A preliminary conspectus of the Allon flora from the Late cretaceous (late Santonian) of central Georgia, USA. Annals of the Missouri Botanical Garden.
Hille, B. (2001) Ion channels of excitable membranes, 3rd edition. Sinauer Associates, Inc.
Hirsch, A.M. (1992) Developmental biology of legume nodulation. New Phytologist, 122, 211-237.
Hodge, A., Campbell, C.D. and Fitter, A.H. (2001) An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature, 413, 297-299.
Hodgkin, A.L. and Katz, B. (1949) The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol., 108, 37-77.
Hohnjec, N., Vieweg, M.F., Puhler, A., Becker, A. and Kuster, H. (2005) Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza. Plant Physiol, 137, 1283-1301.
Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 77, 61-68.
Iida, H., Nakamura, H., Ono, T., Okumura, M.S. and Anraku, Y. (1994) MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating. Mol Cell Biol, 14, 8259-8271.
Imaizumi-Anraku, H., Takeda, N., Charpentier, M., Perry, J., Miwa, H., Umehara, Y., Kouchi, H., Murakami, Y., Mulder, L., Vickers, K., Pike, J., Downie, J.A., Wang, T., Sato, S., Asamizu, E., Tabata, S., Yoshikawa, M., Murooka, Y., Wu, G.J., Kawaguchi, M., Kawasaki, S., Parniske, M. and Hayashi, M. (2005) Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. Nature, 433, 527-531.
Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M. and Sakaki, Y. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A, 98, 4569-4574.
Javot, H., Penmetsa, R.V., Terzaghi, N., Cook, D.R. and Harrison, M.J. (2007) A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci U S A, 104, 1720-1725.
Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B.T. and MacKinnon, R. (2002a) Crystal structure and mechanism of a calcium-gated potassium channel. Nature, 417, 515-522.
92
Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B.T. and MacKinnon, R. (2002b) The open pore conformation of potassium channels. Nature, 417, 523-526.
Journet, E.P., El-Gachtouli, N., Vernoud, V., de Billy, F., Pichon, M., Dedieu, A., Arnould, C., Morandi, D., Barker, D.G. and Gianinazzi-Pearson, V. (2001) Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells. Mol Plant Microbe Interact, 14, 737-748.
Juge, C., Samson, J., Bastien, C., Vierheilig, H., Coughlan, A. and Piche, Y. (2002) Breaking dormancy is spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period. Mycorrhiza, 12, 37-42.
Kaiser, B.N., Finnegan, P.M., Tyerman, S.D., Whitehead, L.F., Bergersen, F.J., Day, D.A. and Udvardi, M.K. (1998) Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules. Science, 281, 1202-1206.
Kanamori, N., Madsen, L.H., Radutoiu, S., Frantescu, M., Quistgaard, E.M., Miwa, H., Downie, J.A., James, E.K., Felle, H.H., Haaning, L.L., Jensen, T.H., Sato, S., Nakamura, Y., Tabata, S., Sandal, N. and Stougaard, J. (2006) A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc Natl Acad Sci U S A, 103, 359-364.
Karimi, M., Inze, D. and Depicker, A. (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci, 7, 193-195.
Kawaguchi, M., Motomura, T., Imaizumi-Anraku, H., Akao, S. and Kawasaki, S. (2001) Providing the basis for genomics in Lotus japonicus: the accessions Miyakojima and Gifu are appropriate crossing partners for genetic analyses. Mol Genet Genomics, 266, 157-166.
Kawasaki, S. and Murakami, Y. (2000) Genome Analysis of Lotus japonicus. J Plant Res, 113, 497-506.
King, M.C., Lusk, C.P. and Blobel, G. (2006) Karyopherin-mediated import of integral inner nuclear membrane proteins. Nature, 442, 1003-1007.
Kistner, C. and Parniske, M. (2002) Evolution of signal transduction in intracellular symbiosis. Trends Plant Sci, 7, 511-518.
Kistner, C., Winzer, T., Pitzschke, A., Mulder, L., Sato, S., Kaneko, T., Tabata, S., Sandal, N., Stougaard, J., Webb, K.J., Szczyglowski, K. and Parniske, M. (2005) Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis. Plant Cell, 17, 2217-2229.
Kolacna, L., Zimmermannova, O., Hasenbrink, G., Schwarzer, S., Ludwig, J., Lichtenberg-Frate, H. and Sychrova, H. (2005) New phenotypes of functional expression of the mKir2.1 channel in potassium efflux-deficient Saccharomyces cerevisiae strains. Yeast, 22, 1315-1323.
Koncz, C. and Schell, J. (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet., 204, 383-396.
Kosuta, S., Chabaud, M., Lougnon, G., Gough, C., Denarie, J., Barker, D.G. and Becard, G. (2003) A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula. Plant Physiol, 131, 952-962.
93
Kosuta, S., Hazledine, S., Sun, J., Miwa, H., Morris, R.J., Downie, J.A. and Oldroyd, G.E. (2008) Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes. Proc Natl Acad Sci U S A, 105, 9823-9828.
Kosuta, S., Winzer, T. and Parniske, M. (2005) Arbuscular mycorrhiza. In Márquez, A.J. (ed.), Lotus japonicus handbook. Springer, pp. 87-95.
Kurdjian, A.C. (1995) Role of the differentiation of root epidermal cells in Nod factor (from Rhizobium meliloti)-induced root-hair depolarization of Medicago sativa. Plant Physiol, 107, 783-790.
LaRue, T. and Weeden, N. (1994) The symbiosis genes of host. Officina Press. Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A DNA transformation-competent
Arabidopsis genomic library in Agrobacterium. Biotechnology (N Y), 9, 963-967. Leckie, C.P., McAinsh, M.R., Allen, G.J., Sanders, D. and Hetherington, A.M. (1998)
Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose. Proc Natl Acad Sci U S A, 95, 15837-15842.
Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G., Prome, J.C. and Denarie, J. (1990) Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature, 344, 781-784.
Levy, J., Bres, C., Geurts, R., Chalhoub, B., Kulikova, O., Duc, G., Journet, E.P., Ane, J.M., Lauber, E., Bisseling, T., Denarie, J., Rosenberg, C. and Debelle, F. (2004) A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses. Science, 303, 1361-1364.
Li, M., Martin, S.J., Bruno, V.M., Mitchell, A.P. and Davis, D.A. (2004) Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs. Eukaryot Cell, 3, 741-751.
Li, Y., Berke, I., Chen, L. and Jiang, Y. (2007) Gating and inward rectifying properties of the MthK K+ channel with and without the gating ring. J Gen Physiol, 129, 109-120.
Lohse, S., Schliemann, W., Ammer, C., Kopka, J., Strack, D. and Fester, T. (2005) Organization and metabolism of plastids and mitochondria in arbuscular mycorrhizal roots of Medicago truncatula. Plant Physiol, 139, 329-340.
Lombardo, F., Heckmann, A.B., Miwa, H., Perry, J.A., Yano, K., Hayashi, M., Parniske, M., Wang, T.L. and Downie, J.A. (2006) Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth. Mol Plant Microbe Interact, 19, 1444-1450.
Long, S.R. (1996) Rhizobium symbiosis: nod factors in perspective. Plant Cell, 8, 1885-1898.
Lu, Z. and MacKinnon, R. (1994) Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel. Nature, 371, 243-246.
Lucocq, J. (1994) Quantitation of gold labelling and antigens in immunolabelled ultrathin sections. J Anat, 184 (Pt 1), 1-13.
Madsen, E.B., Madsen, L.H., Radutoiu, S., Olbryt, M., Rakwalska, M., Szczyglowski, K., Sato, S., Kaneko, T., Tabata, S., Sandal, N. and Stougaard, J. (2003) A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature, 425, 637-640.
Maekawa, T., Kusakabe, M., Shimoda, Y., Sato, S., Tabata, S., Murooka, Y. and Hayashi, M. (2008) Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus. Mol Plant Microbe Interact, 21, 375-382.
94
Marchetti, C., Jouy, N., Leroy-Martin, B., Defossez, A., Formstecher, P. and Marchetti, P. (2004) Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility. Hum Reprod, 19, 2267-2276.
Maresova, L. and Sychrova, H. (2005) Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants. Mol Microbiol, 55, 588-600.
Martens, J.R., Kwak, Y.G. and Tamkun, M.M. (1999) Modulation of Kv channel alpha/beta subunit interactions. Trends Cardiovasc Med, 9, 253-258.
Marvel, D.J., Torrey, J.G. and Ausubel, F.M. (1987) Rhizobium symbiotic genes required for nodulation of legume and nonlegume hosts. Proc Natl Acad Sci U S A, 84, 1319-1323.
Matzke, M.A. and Matzke, A.J.M. (1986) Visualization of mitochondria and nuclei in living plant cells by the use of a potential-sensitive fluorescent dye. Plant, Cell and Environment, 9, 73-77.
McAinsh, M.R., Webb, A., Taylor, J.E. and Hetherington, A.M. (1995) Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium. Plant Cell, 7, 1207-1219.
McDonald, T.V., Yu, Z., Ming, Z., Palma, E., Meyers, M.B., Wang, K.W., Goldstein, S.A. and Fishman, G.I. (1997) A minK-HERG complex regulates the cardiac potassium current I(Kr). Nature, 388, 289-292.
Messinese, E., Mun, J.H., Yeun, L.H., Jayaraman, D., Rouge, P., Barre, A., Lougnon, G., Schornack, S., Bono, J.J., Cook, D.R. and Ane, J.M. (2007) A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula. Mol Plant Microbe Interact, 20, 912-921.
Metivier, D., Dallaporta, B., Zamzami, N., Larochette, N., Susin, S.A., Marzo, I. and Kroemer, G. (1998) Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes. Immunol Lett, 61, 157-163.
Michaelevski, I., Chikvashvili, D., Tsuk, S., Fili, O., Lohse, M.J., Singer-Lahat, D. and Lotan, I. (2002) Modulation of a brain voltage-gated K+ channel by syntaxin 1A requires the physical interaction of Gbetagamma with the channel. J Biol Chem, 277, 34909-34917.
Miles, G.P., Samuel, M.A., Jones, A.M. and Ellis, B.E. (2004) Mastoparan rapidly activates plant MAP kinase signaling independent of heterotrimeric G proteins. Plant Physiol, 134, 1332-1336.
Mitra, R.M., Gleason, C.A., Edwards, A., Hadfield, J., Downie, J.A., Oldroyd, G.E. and Long, S.R. (2004) A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning. Proc Natl Acad Sci U S A, 101, 4701-4705.
Miwa, H., Sun, J., Oldroyd, G.E. and Downie, J.A. (2006) Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus. Mol Plant Microbe Interact, 19, 914-923.
Molokanova, E., Savchenko, A. and Kramer, R.H. (2000) Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein. J Gen Physiol, 115, 685-696.
Morera, F.J., Vargas, G., Gonzalez, C., Rosenmann, E. and Latorre, R. (2007) Ion-channel reconstitution. Methods Mol Biol, 400, 571-585.
95
Murphy, P.J., Langridge, P. and Smith, S.E. (1997) Cloning plant genes differentially expressed during colonization of roots of Hordeum vulgare by the vesicular arbuscular mycorrhizal fungus Glomus intraradices. New Phytologist, 135, 291-301.
Murray, J., Karas, B., Ross, L., Brachmann, A., Wagg, C., Geil, R., Perry, J., Nowakowski, K., MacGillivary, M., Held, M., Stougaard, J., Peterson, L., Parniske, M. and Szczyglowski, K. (2006) Genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype. Mol Plant Microbe Interact, 19, 1082-1091.
Nagata, T., Iizumi, S., Satoh, K., Ooka, H., Kawai, J., Carninci, P., Hayashizaki, Y., Otomo, Y., Murakami, K., Matsubara, K. and Kikuchi, S. (2004) Comparative analysis of plant and animal calcium signal transduction element using plant full-length cDNA data. Mol Biol Evol, 21, 1855-1870.
Nair, M.G., Safir, G.R. and Siqueira, J.O. (1991) Isolation and Identification of Vesicular-Arbuscular Mycorrhiza-Stimulatory Compounds from Clover (Trifolium repens) Roots. Appl Environ Microbiol, 57, 434-439.
Nakamura, Y., Kaneko, T., Asamizu, E., Kato, T., Sato, S. and Tabata, S. (2002) Structural analysis of a Lotus japonicus genome. II. Sequence features and mapping of sixty-five TAC clones which cover the 6.5-mb regions of the genome. DNA Res, 9, 63-70.
Napoli, C.A. and Hubbell, D.H. (1975) Ultrastructure of Rhizobium-induced infection threads in clover root hairs. Appl Microbiol, 30, 1003-1009.
Navazio, L., Bewell, M.A., Siddiqua, A., Dickinson, G.D., Galione, A. and Sanders, D. (2000) Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate. Proc Natl Acad Sci U S A, 97, 8693-8698.
Navazio, L., Mariani, P. and Sanders, D. (2001) Mobilization of Ca2+ by cyclic ADP-ribose from the endoplasmic reticulum of cauliflower florets. Plant Physiol, 125, 2129-2138.
Navazio, L., Moscatiello, R., Genre, A., Novero, M., Baldan, B., Bonfante, P. and Mariani, P. (2007) A diffusible signal from arbuscular mycorrhizal fungi elicits a transient cytosolic calcium elevation in host plant cells. Plant Physiol, 144, 673-681.
Niemietz, C.M. and Tyerman, S.D. (2000) Channel-mediated permeation of ammonia gas through the peribacteroid membrane of soybean nodules. FEBS Lett, 465, 110-114.
Novero, M., Faccio, A., Genre, A., Stougaard, J., Webb, J., Mulder, L., Parniske, M. and Bonfante, P. (2002) Dual requirement of the LjSym4 gene for mycorrhizal development in epidermal and cortical cells of Lotus japonicus roots. New Phytologist, 154, 741-749.
Ohba, T., Schirmer, E.C., Nishimoto, T. and Gerace, L. (2004) Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore. J Cell Biol, 167, 1051-1062.
Oke, V. and Long, S.R. (1999) Bacteroid formation in the Rhizobium-legume symbiosis. Curr Opin Microbiol, 2, 641-646.
Olah, B., Briere, C., Becard, G., Denarie, J. and Gough, C. (2005) Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root
96
formation in Medicago truncatula via the DMI1/DMI2 signalling pathway. Plant J, 44, 195-207.
Oldroyd, G.E. and Downie, J.A. (2006) Nuclear calcium changes at the core of symbiosis signalling. Curr Opin Plant Biol, 9, 351-357.
Oldroyd, G.E., Mitra, R.M., Wais, R.J. and Long, S.R. (2001) Evidence for structurally specific negative feedback in the Nod factor signal transduction pathway. Plant J, 28, 191-199.
Ovtsyna, A.O., Dolgikh, E.A., Kilanova, A.S., Tsyganov, V.E., Borisov, A.Y., Tikhonovich, I.A. and Staehelin, C. (2005) Nod factors induce nod factor cleaving enzymes in pea roots. Genetic and pharmacological approaches indicate different activation mechanisms. Plant Physiol, 139, 1051-1064.
Paidhungat, M. and Garrett, S. (1997) A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(Ts) growth defect. Mol Cell Biol, 17, 6339-6347.
Parniske, M. (2004) Molecular genetics of the arbuscular mycorrhizal symbiosis. Curr Opin Plant Biol, 7, 414-421.
Pearson, J.N. and Jakobsen, I. (1993) The relative contribution of hyphae and roots to phosphorus uptake by arbuscular mycorrhizal plants, measured by dual labelling with 32P and 33P. New Phytologist, 124, 489-494.
Peck, J.W., Bowden, E.T. and Burbelo, P.D. (2004) Structure and function of human Vps20 and Snf7 proteins. Biochem J, 377, 693-700.
Peiter, E., Fischer, M., Sidaway, K., Roberts, S.K. and Sanders, D. (2005) The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity. FEBS Lett, 579, 5697-5703.
Perry, J.A., Wang, T.L., Welham, T.J., Gardner, S., Pike, J.M., Yoshida, S. and Parniske, M. (2003) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol, 131, 866-871.
Pingret, J.L., Journet, E.P. and Barker, D.G. (1998) Rhizobium nod factor signaling. Evidence for a g protein-mediated transduction mechanism. Plant Cell, 10, 659-672.
Poulsen, C. and Podenphant, L. (2002) Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti. Mol Plant Microbe Interact, 15, 376-379.
Radutoiu, S., Madsen, L.H., Madsen, E.B., Felle, H.H., Umehara, Y., Gronlund, M., Sato, S., Nakamura, Y., Tabata, S., Sandal, N. and Stougaard, J. (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature, 425, 585-592.
Radutoiu, S., Madsen, L.H., Madsen, E.B., Jurkiewicz, A., Fukai, E., Quistgaard, E.M., Albrektsen, A.S., James, E.K., Thirup, S. and Stougaard, J. (2007) LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range. Embo J, 26, 3923-3935.
Remy, W., Taylor, T.N., Hass, H. and Kerp, H. (1994) Four hundred-million-year-old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci U S A, 91, 11841-11843.
Ridge, R.W. and Rolfe, B.G. (1985) Rhizobium sp. Degradation of Legume Root Hair Cell Wall at the Site of Infection Thread Origin. Appl Environ Microbiol, 50, 717-720.
97
Riely, B.K., Lougnon, G., Ane, J.M. and Cook, D.R. (2007) The symbiotic ion channel homolog DMI1 is localized in the nuclear membrane of Medicago truncatula roots. Plant J, 49, 208-216.
Robertson, J.G., Wells, B., Brewin, N.J., Wood, E., Knight, C.D. and Downie, J.A. (1985) The legume-Rhizobium symbiosis: a cell surface interaction. J Cell Sci Suppl, 2, 317-331.
Rodriguez-Navarro, A. (2000) Potassium transport in fungi and plants. Biochim Biophys Acta, 1469, 1-30.
Roeper, J., Lorra, C. and Pongs, O. (1997) Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase. J Neurosci, 17, 3379-3391.
Ross, E.M. and Higashijima, T. (1994) Regulation of G-protein activation by mastoparans and other cationic peptides. Methods Enzymol, 237, 26-37.
Saito, K., Yoshikawa, M., Yano, K., Miwa, H., Uchida, H., Asamizu, E., Sato, S., Tabata, S., Imaizumi-Anraku, H., Umehara, Y., Kouchi, H., Murooka, Y., Szczyglowski, K., Downie, J.A., Parniske, M., Hayashi, M. and Kawaguchi, M. (2007) NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus. Plant Cell, 19, 610-624.
Sambrook, J. and Russell, D.W. (2001) Molecular cloning a laboratory manual, 3rd edition. Cold spring harbor laboratory press.
Sandal, N., Krusell, L., Radutoiu, S., Olbryt, M., Pedrosa, A., Stracke, S., Sato, S., Kato, T., Tabata, S., Parniske, M., Bachmair, A., Ketelsen, T. and Stougaard, J. (2002) A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci. Genetics, 161, 1673-1683.
Sandal, N., Petersen, T.R., Murray, J., Umehara, Y., Karas, B., Yano, K., Kumagai, H., Yoshikawa, M., Saito, K., Hayashi, M., Murakami, Y., Wang, X., Hakoyama, T., Imaizumi-Anraku, H., Sato, S., Kato, T., Chen, W., Hossain, M.S., Shibata, S., Wang, T.L., Yokota, K., Larsen, K., Kanamori, N., Madsen, E., Radutoiu, S., Madsen, L.H., Radu, T.G., Krusell, L., Ooki, Y., Banba, M., Betti, M., Rispail, N., Skot, L., Tuck, E., Perry, J., Yoshida, S., Vickers, K., Pike, J., Mulder, L., Charpentier, M., Muller, J., Ohtomo, R., Kojima, T., Ando, S., Marquez, A.J., Gresshoff, P.M., Harada, K., Webb, J., Hata, S., Suganuma, N., Kouchi, H., Kawasaki, S., Tabata, S., Hayashi, M., Parniske, M., Szczyglowski, K., Kawaguchi, M. and Stougaard, J. (2006) Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci. Mol Plant Microbe Interact, 19, 80-91.
Schauser, L., Handberg, K., Sandal, N., Stiller, J., Thykjaer, T., Pajuelo, E., Nielsen, A. and Stougaard, J. (1998) Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus. Mol Gen Genet, 259, 414-423.
Schlosser, A., Hamann, A., Bossemeyer, D., Schneider, E. and Bakker, E.P. (1993) NAD+ binding to the Escherichia coli K(+)-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport. Mol Microbiol, 9, 533-543.
Schüssler, A., Schwarzott, D., Walker, C. (2001) A new fungal phylum, the Glomeromycota phylogeny and evolution. Mycol. Res., 1413-1421.
98
Scott, V.E., Rettig, J., Parcej, D.N., Keen, J.N., Findlay, J.B., Pongs, O. and Dolly, J.O. (1994) Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K+ channels from bovine brain. Proc Natl Acad Sci U S A, 91, 1637-1641.
Shaw, S.L. and Long, S.R. (2003) Nod factor elicits two separable calcium responses in Medicago truncatula root hair cells. Plant Physiol, 131, 976-984.
Shi, J., Blundell, T.L. and Mizuguchi, K. (2001) FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J Mol Biol, 310, 243-257.
Smith, S.E., Smith, F.A. and Jakobsen, I. (2003) Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. Plant Physiol, 133, 16-20.
Soltis, D.E., Soltis, P.S., Chase, M.W., Mort, M.E., Albach, D.C., Zanis, M., Savolainen, V., Hahn, W.H., Hoot, S.B., Fay, M.F., Axtell, M., Swensen, S.M., Prince, L.M., Kress, W.J.O.H.N., Nixon, K.C. and Farris, J.S. (2000) Angiosperm phylogeny inferred from 18S rDNA, rbcL, and atpB sequences. Botanical Journal of the Linnean Society.
Spaink, H.P., Sheeley, D.M., van Brussel, A.A., Glushka, J., York, W.S., Tak, T., Geiger, O., Kennedy, E.P., Reinhold, V.N. and Lugtenberg, B.J. (1991) A novel highly unsaturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium. Nature, 354, 125-130.
Stehno-Bittel, L., Lückhoff, A. and Clapham, D.E. (1995) Calcium release from the nucleus by InsP3 receptor channels. Neuron, 14, 163-167.
Stern, J.H., Knutsson, H. and MacLeish, P.R. (1987) Divalent cations directly affect the conductance of excised patches of rod photoreceptor membrane. Science, 236, 1674-1678.
Stougaard, J. (2000) Regulators and regulation of legume root nodule development. Plant Physiol, 124, 531-540.
Stougaard, J., Abildsten, D. and KA, M. (1987a) The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Molecular General Genetics, 207, 251-255.
Stougaard, J., Abildsten, D. and Marker, K. (1987b) The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Molecular General Genetics, 207, 251-255.
Stracke, S., Kistner, C., Yoshida, S., Mulder, L., Sato, S., Kaneko, T., Tabata, S., Sandal, N., Stougaard, J., Szczyglowski, K. and Parniske, M. (2002) A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature, 417, 959-962.
Sullivan, J.T., Patrick, H.N., Lowther, W.L. and Scott, D.B. (1995) Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment. PNAS, 92, 8985-8989.
Sun, J., Miwa, H., Downie, J.A. and Oldroyd, G.E. (2007) Mastoparan activates calcium spiking analogous to Nod factor-induced responses in Medicago truncatula root hair cells. Plant Physiol, 144, 695-702.
Sutton, J.M., Lea, E.J. and Downie, J.A. (1994) The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes. Proc Natl Acad Sci U S A, 91, 9990-9994.
Swensen, S.M. and Benson, D.R. (2008) Evolution of actinorhizal host plants and Frankia endosymbionts. In Pawlowski, K. and Newton, W.E. (eds.), Nitrogen-fixing actinorhizal symbioses. Dordrecht:Springer Netherlands, pp. 73-104.
99
Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T. and et al. (1989) Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature, 339, 439-445.
Tansengco, M.L., Hayashi, M., Kawaguchi, M., Imaizumi-Anraku, H. and Murooka, Y. (2003) crinkle, a novel symbiotic mutant that affects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus. Plant Physiol, 131, 1054-1063.
Tester, M. and Davenport, R. (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot (Lond), 91, 503-527.
Timmers, A.C., Auriac, M.C. and Truchet, G. (1999) Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development, 126, 3617-3628.
Tinker, A., Lindsay, A.R. and Williams, A.J. (1992) Block of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by tetra-alkyl ammonium cations. J Membr Biol, 127, 149-159.
Tirichine, L., Imaizumi-Anraku, H., Yoshida, S., Murakami, Y., Madsen, L.H., Miwa, H., Nakagawa, T., Sandal, N., Albrektsen, A.S., Kawaguchi, M., Downie, A., Sato, S., Tabata, S., Kouchi, H., Parniske, M., Kawasaki, S. and Stougaard, J. (2006) Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development. Nature, 441, 1153-1156.
Trappe, J.M. (1987) Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an evolutionary standpoint.
Truchet, G., Roche, P., Lerouge, P., Vasse, J., Camut, S., de Billy, F., Promé, J.C. and Dénarié, J. (1991) Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature, 351, 670-673.
Tsyganov, V.E., Voroshilova, V.A., Priefer, U.B., Borisov, A.Y. and Tikhonovich, I.A. (2002) Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis. Ann Bot (Lond), 89, 357-366.
Turgeon, B.G. and Bauer, W.D. (1985) Ultrastructure of infection-thread development during infection of soybean by Rhizobium japonicum. Planta, 163, 328-349.
Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., Kyozuka, J. and Yamaguchi, S. (2008) Inhibition of shoot branching by new terpenoid plant hormones. Nature, 455, 195-200.
Valiyaveetil, F.I., Leonetti, M., Muir, T.W. and Mackinnon, R. (2006) Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation. Science, 314, 1004-1007.
van Brussel, A.A., Bakhuizen, R., van Spronsen, P.C., Spaink, H.P., Tak, T., Lugtenberg, B.J. and Kijne, J.W. (1992) Induction of Pre-Infection Thread Structures in the Leguminous Host Plant by Mitogenic Lipo-Oligosaccharides of Rhizobium. Science, 257, 70-72.
Vierheilig, H., Bago, B., Albrecht, C., Poulin, M.J. and Piche, Y. (1998) Flavonoids and arbuscular-mycorrhizal fungi. Adv Exp Med Biol, 439, 9-33.
Villarejo, A., Buren, S., Larsson, S., Dejardin, A., Monne, M., Rudhe, C., Karlsson, J., Jansson, S., Lerouge, P., Rolland, N., von Heijne, G., Grebe, M., Bako, L. and
100
Samuelsson, G. (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat Cell Biol, 7, 1224-1231.
Wais, R.J., Galera, C., Oldroyd, G., Catoira, R., Penmetsa, R.V., Cook, D., Gough, C., Denarie, J. and Long, S.R. (2000) Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. Proc Natl Acad Sci U S A, 97, 13407-13412.
Walker, D.J., Leigh, R.A. and Miller, A.J. (1996) Potassium homeostasis in vacuolate plant cells. Proc Natl Acad Sci U S A, 93, 10510-10514.
Walker, S.A. and Downie, J.A. (2000) Entry of Rhizobium leguminosarum bv.viciae into root hairs requires minimal Nod factor specificity, but subsequent infection thread growth requires nodO or nodE. Mol Plant Microbe Interact, 13, 754-762.
Walker, S.A., Viprey, V. and Downie, J.A. (2000) Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by nod factors and chitin oligomers. Proc Natl Acad Sci U S A, 97, 13413-13418.
Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K. and Kudla, J. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J, 40, 428-438.
Wang, J. and Best, P.M. (1994) Characterization of the potassium channel from frog skeletal muscle sarcoplasmic reticulum membrane. J Physiol, 477 (Pt 2), 279-290.
Weidmann, S., Sanchez, L., Descombin, J., Chatagnier, O., Gianinazzi, S. and Gianinazzi-Pearson, V. (2004) Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula. Mol Plant Microbe Interact, 17, 1385-1393.
Wilkinson, D.M. (2001) At cross purposes. Nature, 412, 485. Winter, V. and Hauser, M.T. (2006) Exploring the ESCRTing machinery in eukaryotes.
Trends Plant Sci, 11, 115-123. Wydro, M., Kozubek, E. and Lehmann, P. (2006) Optimization of transient
Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana. Acta Biochim Pol, 53, 289-298.
Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L. and Wu, W.H. (2006) A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell, 125, 1347-1360.
Xu, W. and Mitchell, A.P. (2001) Yeast PalA/AIP1/Alix homolog Rim20p associates with a PEST-like region and is required for its proteolytic cleavage. J Bacteriol, 183, 6917-6923.
Xu, W., Smith, F.J., Jr., Subaran, R. and Mitchell, A.P. (2004) Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans. Mol Biol Cell, 15, 5528-5537.
Yang, W.C., de Blank, C., Meskiene, I., Hirt, H., Bakker, J., van Kammen, A., Franssen, H. and Bisseling, T. (1994) Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation. Plant Cell, 6, 1415-1426.
Yano, K., Tansengco, M.L., Hio, T., Higashi, K., Murooka, Y., Imaizumi-Anraku, H., Kawaguchi, M. and Hayashi, M. (2006) New nodulation mutants responsible for infection thread development in Lotus japonicus. Mol Plant Microbe Interact, 19, 801-810.
101
Yao, P.Y. and Vincent, J.M. (1969) Host specificity in the root hair "curling factor" of Rhizobium spp. Aust J Biol Sci, 22, 413-423.
Yellen, G. (1984) Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. J Gen Physiol, 84, 157-186.
Yoshida, S. and Parniske, M. (2005) Regulation of plant symbiosis receptor kinase through serine and threonine phosphorylation. J Biol Chem, 280, 9203-9209.
Zadek, B. and Nimigean, C.M. (2006) Calcium-dependent gating of MthK, a prokaryotic potassium channel. J Gen Physiol, 127, 673-685.
Zhang, Y., Niu, X., Brelidze, T.I. and Magleby, K.L. (2006) Ring of negative charge in BK channels facilitates block by intracellular Mg2+ and polyamines through electrostatics. J Gen Physiol, 128, 185-202.
Zhu, H., Riely, B.K., Burns, N.J. and Ane, J.M. (2006) Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics, 172, 2491-2499.
Zimmermann, S., Nurnberger, T., Frachisse, J.M., Wirtz, W., Guern, J., Hedrich, R. and Scheel, D. (1997) Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense. Proc Natl Acad Sci U S A, 94, 2751-2755.
102
6. Acknowledgement
I would like to express my gratitude to all those who gave me the possibility to complete
this thesis. First, I want to thank my supervisor Prof. Dr. M. Parniske for giving me the
chance to perform my PhD-thesis in his laboratory, for supporting my work and giving
me the opportunity to have a worldwide scientific training.
My colleagues past and present all gave me the feeling to be at home at work. Therefore,
I like to thank all members of the laboratory for buidling a great atmosphere.
I like also to give a special thanks to Lionel Navarro, Cristina Azevedo, Gabor
Giczey, Satoko Yoshida, Catherine White and Meritxell Llovera for stimulating
discussions, suggestions and encouragement, which helped me in research for this thesis.
For all their help, interest and valuable hints, I like to specially thank Rolf Bredemeier
and Prof. Dr. Enrico Schleiff. Furthermore, I like to express my sincere thanks to my PhD
colleagues, Kristina Haage, Martin Groth and Katharina Markmann for all their help in
running the laboratory and their support in a foreign country.
My sincere thanks are also due to all my friends in Munich and family in France for their
patience, support, interest, and encouragement in difficult time. I warmly thank my
friends Marie and Benoit, as well as my brother for their understanding and
encouragement all time of my research. I would like to give my special thanks to René
for patient and loving support enabled me to complete this work.
103
7. Appendix
7.1 List of publications
7.1.1 Papers
Perry, J., Welham, T., Brachmann, A., Charpentier, M., Markmann, K., Wang, T. and Parniske, M. Mining the symbiotic component of the Lotus japonicus genome using classical genetics and thematic TILLING. Manuscript in preparation.
Charpentier, M., Bredemeier, R., Wanner, G., Takeda, N., Schleiff, E. and Parniske, M. Lotus japonicus CASTOR and POLLUX Are Ion Channels Essential for Perinuclear Calcium Spiking in Legume Root Endosymbiosis. Plant Cell, 20, 1-13
Sandal, N., Petersen, T.R., Murray, J., Umehara, Y., Karas, B., Yano, K., Kumagai, H., Yoshikawa, M., Saito, K., Hayashi, M., Murakami, Y., Wang, X., Hakoyama, T., Imaizumi-Anraku, H., Sato, S., Kato, T., Chen, W., Hossain, M.S., Shibata, S., Wang, T.L., Yokota, K., Larsen, K., Kanamori, N., Madsen, E., Radutoiu, S., Madsen, L.H., Radu, T.G., Krusell, L., Ooki, Y., Banba, M., Betti, M., Rispail, N., Skot, L., Tuck, E., Perry, J., Yoshida, S., Vickers, K., Pike, J., Mulder, L., Charpentier, M., Muller, J., Ohtomo, R., Kojima, T., Ando, S., Marquez, A.J., Gresshoff, P.M., Harada, K., Webb, J., Hata, S., Suganuma, N., Kouchi, H., Kawasaki, S., Tabata, S., Hayashi, M., Parniske, M., Szczyglowski, K., Kawaguchi, M. and Stougaard, J. (2006) Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci. Mol Plant Microbe Interact, 19, 80-91
Imaizumi-Anraku, H., Takeda, N., Charpentier, M., Perry, J., Miwa, H., Umehara, Y., Kouchi, H., Murakami, Y., Mulder, L., Vickers, K., Pike, J., Downie, J.A., Wang, T., Sato, S., Asamizu, E., Tabata, S., Yoshikawa, M., Murooka, Y., Wu, G.J., Kawaguchi, M., Kawasaki, S., Parniske, M. and Hayashi, M. (2005) Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. Nature, 433, 527-531.
7.1.2 Posters and conferences
CASTOR and POLLUX in symbiotic signal transduction. Charpentier, M., Bredemeier, R., Schleiff, E. and Parniske, P. International Symposium: 100 years of Endosymbiotic Theory: Form Procaryotes to Eucaryotic Organelles. Hamburg, Germany (2005).
104
CASTOR and POLLUX in symbiotic signal transduction. Charpentier, M., Bredemeier, R., Schleiff, E. and Parniske, P. Gordon conference: Ion channels. Tilton, NH, USA (2006). CASTOR and POLLUX ion transporters in symbiotic signal transduction: myth or reality? Charpentier, M., Bredemeier, R., Schleiff, E. and Parniske, P. XV FESPB Congress. Lyon, France (2006)
CASTOR and POLLUX in symbiotic signal transduction. Charpentier, M., Bredemeier, R., Schleiff, E. and Parniske, P. 13th International congress on Molecular Plant-Microbe Interaction. Sorrento, Italy (2007)
7.1.3 Talks
CASTOR and POLLUX in symbiotic signal transduction. Intensifying Training in Europe on Genomic Research Activity in Legumes network (INTEGRAL) Conference. Munich, Germany (2005)
CASTOR and POLLUX in symbiotic signal transduction. INTEGRAL Conference. Ravello, Italia (2006)
Unravelling the function of CASTOR and POLLUX in symbiotic signalling. INTEGRAL Conference. Sevilla, Spain (2007)
New class of cation channels essential for perinuclear calcium spiking. 8th European Nitrogen Fixation Conference. Gent, Belgium (2008)
105
7.2 List of figures
Figure 1. Positional cloning of CASTOR and POLLUX genes……………………………. 25 Figure 2. A model for the early symbiosis signalling pathway in the epidermis…………..26 Figure 3. Alternative-spliced variants of CASTOR (A) and POLLUX (B)………………... 28 Figure 4. Unrooted radial phylogenetic tree of CASTOR and POLLUX homologs……… 32 Figure 5. Sequence analyses of CASTOR and POLLUX…………………………………. 34 Figure 6. Localization in tobacco leaf epidermal cells of CASTOR and POLLUX………. 36 Figure 7. Immunogold localization of CASTOR in Lotus japonicus with anti-CASTOR... 39 Figure 8. Yeast-two-hybrid assay for interactions of cCASTOR and cPOLLUX………… 41 Figure 9. Formation of CASTOR and POLLUX homocomplexes in planta……………... 42 Figure 10. Expression patterns of CASTOR and POLLUX promoter:GUS fusions in L. japonicus roots before and after inoculation with Mesorhizobium loti………………… 44 Figure 11. Restoration of root nodule symbiosis in A. rhizogenes-transformed root systems of L. japonicus mutants…………………………………………………………………….. 45 Figure 12. Restoration of root nodule symbiosis in A. rhizogenes-transformed root systems of L. japonicus mutant with the M. truncatula DMI1 gene under the control of single P35S…………………………………………………………………………………. 46 Figure 13. Complementation of the yeast double mutant cch1∆mid1∆ and trk1∆ strains.... 48 Figure 14. Complementation of MAB 2d mutant…………………………………………. 49 Figure 15. Cell free expression of CASTOR, castor-2 and POLLUX…………………….. 51 Figure 16. Electrophysiological characterization of CASTOR……………………………. 52
Figure 17. Modelization of the CASTOR pore and the castor-2 mutant pore…………….. 53 Figure 18. Electrophysiological characterization of castor-2 channel…………………….. 53 Figure 19. Voltage dependent magnesium-blockage of CASTOR………………………... 55 Figure 20. Modulation of CASTOR gating……………………………………………….. 56
106
Figure 21. Yeast-two-hybrid assay for interaction between cCASTOR and yeast two hybrid library full-length candidat interactors………………………………………... 58 Figure 22. Yeast-two-hybrid assay for interactions of cCASTOR and yeast two hybrid library full length candidate interactors……………………………………………. 61 Figure 23. Downregulation of LjSNF7 in A. rhizogenes transformed roots expressing pUB-GWS50-GFP impaired nodulation………………………………………. 62 Figure 24. Subcellular localization of LjSNF7:RFP in L. japonicus cell culture protoplast…………………………………………………………………………... 63 Figure 25. Unrooted radial phylogenetic tree of LjSNF7 homologs……………………… 69 Figure 26. Proposed role of CASTOR and POLLUX as counter ion channels…………… 71 Figure 27. Representation of the fusion of proteoliposome with the planar lipids bilayer... 81 Figure 28. Lipid-bilayer-measurement system……………………………………………. 81 7.3 List of tables Table 1. Common L. japonicus SYM genes required for AM and RNS…………………... 21 Table 2. List of castor mutant alleles……………………………………………………... 29 Table 3. List of pollux mutant alleles………………………………………………………30 Table 4. Restoration of root nodules symbioses in L. japonicus castor-12 and pollux-5 mutants…………………………………………………………………………… 31 Table 5. Restoration of Root Nodule Symbioses in A. rhizogenes-transformed root systems of L. japonicus mutants pollux-2 and castor-12 with the different POLLUX and CASTOR split YFP versions, respectively…………………………………………….. 42 Table 6. Reverse potential (Erev) and permeability ratios (PX/PCl-) for CASTOR and castor-2 (A), and reverse potential and permeability ratios PK+/PX for CASTOR (B)……... 52 Table 7. List of putative cCASTOR interactors…………………………………………… 60 Table 8. Construct used in this study……………………………………………………… 74 Table 9. Antibodies used in this study…………………………………………………….. 75
107
7.4 Erklärung
Ich erkläre hiermit an Eides Statt, dass ich die vorgelegte Dissertation über die
Functional Characterisation of Two Channels Proteins
Involved in Leguminous Symbiosis
selbständig angefertigt und mich anderer Hilfsmittel als die in ihr angegebenen nicht
bedient habe, insbesondere, dass aus Schriften Entlehnungen, soweit sie in der
Dissertation nicht ausdrücklich als solche mit Angabe der betreffenden Schrift bezeichnet
sind, nicht stattgefunden haben.
Ich habe weder anderweitig versucht, eine Dissertation einzureichen oder eine
Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile derselben
einer anderen Prüfungskommission vorgelegt.
München, den…………………….. ………………………………
Charpentier Myriam
108
7.5 Curriculum vitae
PERSONAL DATA Date of Birth Feb. 24, 1978. La Roche Sur Yon (Vendée, France) Address 106 Les Cerisiers, 85480 Fougeré. France HIGHER EDUCATION Sept. 1996-June 2000 Institut Catholique d´Etudes Superieures (ICES), La Roche sur yon,
France. Study of Cellular Biology and Physiology
Sept. 2000-June 2001 Paul Sabatier University – Toulouse, France. Master´s degree 1: Physiology and Molecular Plant Biology; (First Class honours). Supervisor: Dr. Georges Boudart (UMR CNRS_UPS 5546) Topic: Elicitor activity of a fungal endopolygalacturonase in tobacco requires a functional catalytic site and cell wall localization
Sept. 2001-Dec. 2002 University Paris VII. France. Master´s degree 2: Plant Productivity; (First Class honours). Supervisor: Dr. Abdel Ihafid Bendahmane (Plant Genomics Research Unit, URGV, Evry) Topic: Map-based cloning of Pmw conferring resistance to Sphaerotheca fulginea in melon
Oct. 2004- Sept. 2008 PhD (Ludwig-Maximilians-Universität München, Genetics: Prof.
Dr. Martin Parniske) Topic: Functional characterization of CASTOR and POLLUX, new
class of potassium permeable channel involved in arbuscular mycorrhiza and root nodule symbioses
MISCELLANEOUS First Aid Training course (France) Computational skills Microsoft Word/ Excel/Power Point/Adobe Photoshop Endnote/Vector NTI/Staden Package/DeepView.
Experience with both Windows and Macintosh operating systems. Languages English (IELTS, 6.5), German (basic knowledge) Hobbies Running; Dancing (salsa); Music (Accordeon)